... estimation of protein. A. Modified Lowry's method ... Af. Phenolphthalein phosphate agar ..... 54 gm Tris base, 27.5 gm boric acid, 20 ml of 0.5 M EDTA (pH 8.0).
Labtop for Microbiology LaboratoryTable of Contents S. No. 1.
Page number 9
6. 7
Title Rates in centrifugation a. Settling rate b. RCF c. Volume and centrifugal force d. Fixed versus swing out rotor Glass ware cleansing solution Common tissue preservatives Trypan blue solution for intravenous use Buffers used for suspending live cells, bacteria and other live materials Buffers/solution for plasmid/chromosomal DNA isolation Solutions for plasmid isolation by alkaline lysis
8.
Buffers/solutions for SDS-PAGE
13-14
9.
Recipes for making discontinuous dissociating gels
14
10.
Recipes for making discontinuous non-dissociating gels
14
11.
Recipes for making continuous non-dissociating gels
14
12.
Gel processing solutions:
15
13.
ELISA-reagents and buffers
15-17
14.
Solutions for estimation of protein
17-18
2. 3. 4. 5.
10 10 10 10-11 11-12 12-13
A. Modified Lowry’s method B. UV spectrophotometery C. Biurate method D. Bicinchoninic acid method E. Dye binding method F. Silver binding method 15.
pH of standard solutions of common chemicals used in 19 laboratory
16.
Physical characteristics of common acids and alkalies 19 used in laboratory
17
Buffers for specific purposes a. Mcilvaine’s buffer 20 b. Phthalate buffer c. PBS, 0.025 m, pH 6.0 and 6.8 d. PBS, 0.15 m (pH 7.0) e. PBS, pH 7.2 f. PBS, pH 7.4 g. PBS, pH 7.3 h. Azide saline, pH 7.3
20-25
1
i. Borate calcium saline, pH 7.3 j. Veronal-NaCl diluent 5x k. Sorensen’s citrate buffer l. Citrate phosphate buffer for pH 2.6 to 7.0 m. Citrate buffer pH 3.0 to 6.2 n. Phosphate buffer for pH 5.7 to 8.0 (0.25m) o. Phosphate buffer for pH 5.8 to 8.0 (0. 5 m) p. Potassium phosphate buffer for pH 5.8 to 8.0 (0. 1 m) q. PBS (bacteriological) r. Tris buffer s. Barbital buffer (0.05 m) pH 6.8 to 9.2 t. Borate buffer (0.0125 m) pH 8.1 to 9.0 u. Borate buffer (0.0125 m) pH 9.3 to 10.7 v. Glycine buffer (0.05 m) pH 8.6 to 10.6 w. Sodium carbonate bicarbonate buffer (0.05 m) pH 9.2 18. 19. 20.
21.
22. 23. 24.
25.
to 10.8 x. Carbonate buffer (0.025 m) pH 9.7 to 10.9 Antibiotic solutions Sugar solutions Dye solutions and indicators A. Common dyes B. Andrade’s indicator C. Litmus solution Common culture media used in microbiology A. Tryptic soy broth B. Semisolid phosphate buffered agar C. Gelatin agar D. Organic acid media E. Decarboxylase test media F. Luria bertani broth G. 1% peptone water H. RPMI-1640 growth medium I. MEM (minimum essential media) J. M-9 agar K. Aro mix Clinical sample transport media Pre-enrichment media and resuscitation media Storage of cultures for future use A. On/in media a. Dorset egg medium b. Buffered semisolid nutrient agar c. Glycerol broth d. Stock culture agar e. In sterilized powder of multani mitti (fullers earth) B. Freezing the cultures C. Freeze-drying Some special media and reagents required in bacteriology A. Media for blood cultures in cases of typhoid
25-26 26 27
28-30
30 31 31-33
33 332 33-35
2
26.
B. Swarm-agar (Guard plate) C. Semi-solid agar in u tubes D. Worfel Ferguson medium for capsule enhancement E. Minimal salt medium F. Glucose minimal salt agar medium G. Minimal agar medium H. Deca strength phage broth (DSPB) Bacteriological media used in biochemical 36-43 characterization of bacteria A. MR-VP test medium B. Dextran and levan production medium C. Sucrose broth D. Starch agar E. Aesculin broth F. Aesculin blood agar G. Capsulation medium for Bacillus anthracis (serumbicarbonate agar) H. Casein agar I. Christensen’s citrate medium J. Castaneda medium: K. Koser’s citrate medium L. Simmons’ citrate M. Organic acid medium N. Decarboxylase medium O. Hippurate hydrolysis medium P. KCN broth Q. Loeffler’s serum slants R. LJ medium (lowenstein-jensen medium for mycobacteria) S. Lecithinase agar/ lecithinovitellin agar T. Malonate-phenylalanine medium U. Litmus milk V. Purple milk W. Purple milk X. Motility agar Y. Sulphide indole motility agar Z. MRS lactobacillus agar Aa. Nitrate reduction test media Ab. Nitrite medium Ac. O/F test medium Ad. ONPG broth Ae. Sodium potassium magnesium (SPM) broth Af. Phenolphthalein phosphate agar Ag. Phenylalanine agar Ah. Polyhydroxybutyrate agar Ai. Pyruvate fermentation medium Aj. Salt broth Ak Selenite broth Al. Soil extract agar Am. Streptomyces extraction medium for preparing of streptococcal group antigen An. Todd-Hewitt broth
3
27.
28.
29. 30. 31. 32.
Ao. Media for antigen extraction from streptococci for grouping Ap. TSI (triple sugar iron) agar Aq. Tyrosine hydrolysis agar Ar. Urea medium a. Christensen’s medium b. Christensen’s broth medium As. Xanthine and hypoxanthine agar Some basic media used in microbiology A. Nutrient broth (NB) B. Nutrient agar C. Semisolid NA D. Peptone water E. PW agar F. Robertson’s cooked meat medium (RCM) G. Brain heart infusion broth (BHI) H. BHI agar I. Mueller Hinton agar J. Blood agar (BA) K. Layered BA L. Chocolate agar M. Serum agar N. Serum glucose agar O. Fildes agar and broth P. Glucose broth Q. Glycerol broth R. Mac-Conkey broth double strength S. Laural tryptose broth, double strength T. Brilliant green lactose bile broth U. Tryptone water V. Membrane lauryl sulphate broth Media for detecting pigment production ability of bacteria A. Chromobacterium B. Mycobacterium C. Pseudomonas D. Serratia marcescens E. Micrococcus spp F. Melaninogenicus oralis G. Clostridium difficile H. Potato slopes I. Mannitol yeast extract agar J. King’s medium for pyocyanin K. King’s medium for fluorescine Mc-Farland standard preparation Some important parameters for experimental animals used in microbiology Sizes of hypodermic needles used in microbiology Precipitation of protein antigens without denaturation A. Salting out with ammonium sulphate B. Precipitation with water miscible organic solvent as ethanol and acetone
43-45
45-46
46 47 47 48
4
33.
34.
35.
36.
C. Precipitation with water miscible organic polymers as polyethylene glycol Precipitation of protein antigens by denaturation A. High temperature B. Extreme pH C. Organic solvents Reagents required for different bacteriological tests A. Phenylalanine test reagent B. Gelatinase and caseinase test reagents C. MR reagent D. Nitrate test reagent E. VP test reagents F. Ehrlich’s reagent for indole test G. Benedict’s reagent for presence of reducing sugars H. Acid ferric chloride for phenylpyruvic acid I. Acid mercuric chloride for proteinases J. Nessler’s reagent Bacteriological test strips impregnated with reagents a. For detection of H2S b. For indole test c. PPA test d. For H2O2 e. For oxidase test f. O/129 discs for Vibrionaceae g. Optochin discs h. Liquoid discs for inhibition of Peptococcus anaerobius & Streptobacillus moniliformis i. X-factor discs j. V-factor discs Common rapid tests for identification of bacteria a. Acetyl-methyl carbinol production test (VP test) b. Aesculin bile test for aesculin hydrolysis by streptococci c. Bile solubility test d. Pseudocatalase e. Coagulase: f. Rapid coccal transformation g. Decarboxylase test h. DNase test i. Gelatinase test j. Hippurate hydrolysis test for enterobacteriaceae k. Indole test l. Gluconate oxidation test m. Gluconate utilization broth n. Malonate utilization o. Niacin (nicotinic acid) test p. ONPG (o-nitrophenyl-β-d-galactopyranoside) test q. Oxidase test (cytochrome oxidase) r. Catalase test s. Phenylalanine test t. Levon/ dextran production test u. Porphyrin test (for determining requirement of factor
48-49
49-50
50-51
52-60
5
X) Phosphatase test Tween hydrolysis Urease test X & V factor requirement test Carbon dioxide requirement for bacterial growth (candle jar) aa. Nitrate reduction test Ab. Temperature tolerance and growth temperature Ac. Co-aggregation test Ad. Carboxymethyl cellulose hydrolysis test Ae. Pyrase test Af. Benzidine test Stains and reagents used in microbiology A. Acetone-iodine solution B. Strong iodine solution C. Acid alcohol D. Albert’s stain E. Ammonical silver nitrate F. Ammonium oxalate crystal violet stain G. Different common dyes. H. Aqueous iodine (BP) I. Liquor iodi fortis (BP) J. Carbol fuchsin K. Giemsa stain stock L. Kirkpatrick’s fixative M. Loeffler’s methylene blue (polychrome methylene blue) N. Lugol’s iodine O. Albert’s iodine P. Iodine acetone decolourizer Q. Weak-iodine acetone decolourizer R. Muir’s mordant S. Plimmer’s mordant T. Ryu’s mordant U. Rhodes’ mordant V. Neisser’s staining solution W. Ryu’s flagella stain X. Sudan black Y. Buffers for dilution of Leishman’s stain and washing of slides Staining methods in microbiology A. Simple staining B. Gram’s staining C. Acid-fast staining (Ziehl Neelsen’s method) D. Auramine-phenol stain for fluorescence method E. Spore staining i. Moeller’s method ii. Schaeffer and Fulton’s method iii. A modified Ziehl-Neelsen staining procedure F. Capsule staining i. Muir’s method ii. Giemsa method v. w. x. y. z.
37.
38.
60-63
63-67
6
39. 40.
41. 42. 43.
44. 45.
46. 47. 48.
49. 50. 51. 52
iii. India-ink method (negative staining) G. Staining of lipid granules i. Holbrook and Anderson method ii. Burdon’s method H. Metachromatic granule (volutin granules) staining i. Albert’s method ii. Neisser’s method I. Flagella staining i. Ryu’s method ii. Cesares-Gill’s method iii. Rhodes’ method J. Fungal staining (for cell polysaccharides) i. Direct microscopy ii. Parker-blue staining iii. PAS (per-iodic acid Schiff) staining K. Staining for spirochetes i. Fontana’s method for films ii. Levaditi’s method for staining spirochetes in tissues DNA staining Fixatives used in microbiology a. Formalin b. Susa’s fixative c. Bouin’s fluid d. Schaudinn’s fluid e. Flemming’s fluid General protocol for embedding the fixed tissues Inhibitors for swarming Sterilization A. Heat B. Chemical C. Radiation D. Filtration Media used for sterility testing Indicator organisms a. Coliforms b. Faecal coliforms c. Faecal E. coli d. Faecal streptococci e. Sulphite reducing clostridia f. Pseudomonas g. Bacteriophages Sampling plan for polluted waters Membrane filtration test for indicator microbes Air sampling A. Settle plate method B. Slit sampler MPN table for indicator bacteria Some important conversion factors Equivalents Index
67 67-68
68 68-69 59-70
70 70-71
71 71-72 72-73
73-77 78 78 79-83
7
Acknowledgements Help rendered in terms of facilities to work by the Indian Council of Agricultural Research, India is thankfully acknowledged. I am thankful for moral support from my wife (Geeta), daughters (Sumedha and Richa) and Dr. BP Bhatt (Joint ICAR Research Complex for NEH Region, Nagaland Centre, Jharnapani).
It is dedicated to my father, who put so many efforts for my education but could not see myself as I am today in his lifetime.
8
Labtop Book for Microbiology1. RATES IN CENTRIFUGATION: a. Settling rate (cm/second)=2a2g(dp-dm)/9η Where, dp=density of the particle, g=981 cms-2, dm=density of medium, η =viscosity (in cgs unit) of medium b. Relative centrifugal force (RCF, in g)= 1.118×10-5×R×N2 g=9.81ms-2, R=radius of the centrifuge rotor in cm, it is distance between center of centrifuge shaft and tip of the tube, N=revolution per min. 500g means 500 times the force of gravity i.e., the particle will settle 500 times faster than at the bench top. RPM versus RCF Conversion Table Speed RPM 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 10500 11000 11500 12000 13000 13500 14000
4 45 101 179 280 402 548 716 906 1118 1353 1610 1889 2191 2516 2862 3231 3622 4036 4472 4930 5411 5914 6440 7558 8150 8765
RCF with Rotor Radius (from center of rotor to sample) in centimeters 5 6 7 8 9 10 11 12 13 56 67 78 89 101 112 123 134 145 126 151 176 201 226 252 277 302 327 224 268 313 358 402 447 492 537 581 349 419 489 559 629 699 769 839 908 503 604 704 805 906 1006 1107 1207 1308 685 822 959 1096 1233 1370 1507 1643 1780 894 1073 1252 1431 1610 1789 1968 2147 2325 1132 1358 1585 1811 2038 2264 2490 2717 2943 1398 1677 1957 2236 2516 2795 3075 3354 3634 1691 2029 2367 2706 3044 3382 3720 4058 4397 2012 2415 2817 3220 3622 4025 4427 4830 5232 2362 2834 3306 3779 4251 4724 5196 5668 6141 2739 3287 3835 4383 4930 5478 6026 6574 7122 3144 3773 4402 5031 5660 6289 6918 7547 8175 3578 4293 5009 5724 6440 7155 7871 8586 9302 4039 4847 5654 6462 7270 8078 8885 9693 10501 4528 5433 6339 7245 8150 9056 9961 10867 11773 5045 6054 7063 8072 9081 10090 11099 12108 13117 5590 6708 7826 8944 10062 11180 12298 13416 14534 6163 7396 8628 9861 11093 12326 13559 14791 16024 6764 8117 9469 10822 12175 13528 14881 16233 17586 7393 8871 10350 11828 13307 14786 16264 17743 19221 8050 9660 11269 12879 14489 16099 17709 19319 20929 9447 11337 13226 15115 17005 18894 20784 22673 24562 10188 12225 14263 16300 18338 20376 22413 24451 26488 10956 13148 15339 17530 19722 21913 24104 26295 28487
14 157 352 626 978 1409 1917 2504 3170 3913 4735 5635 6613 7669 8804 10017 11309 12678 14126 15652 17256 18939 20700 22539 26452 28526 30678
15 168 377 671 1048 1509 2054 2683 3396 4193 5073 6037 7085 8217 9433 10733 12116 13584 15135 16770 18489 20292 22178 24149 28341 30563 32869
c. Volume is important factor to achieve results of centrifugation, it determines success of centrifugation. If using low volume yeast, bacteria and viruses settle at 2,000g, 5,000g and 150,000g, respectively while on using large volumes it needs 5,000g, 20,000g and 200,000g, respectively. d. Vertical rotor needs to be run at low speed and for shorter duration than horizontal/ angled or swing out rotors to achieve the same effect.
9
2. GLASS WARE CLEANSING: Dichromate-Sulfuric acid solution: Dissolve 63 g of sodium or potassium dichromate by heating in 35 ml water. Cool and add concentrate H2SO4 to make volume 1 lt. It is very corrosive and must be handled with utmost care and if splashes come in contact of cloths or skin wash with plenty of water and neutralize with sodium bicarbonate solution. The dirty glass items can be directly dipped into it for 1 day or more and then cleaned with water.
3. COMMON TISSUE PRESERVING SOLUTION: Formal saline solution (10.0% v/v): Used for preservation of pathological specimen and also to kill bacteria. For killing bacteria 1% formalin is sufficient to preserve the surface antigens of most bacteria. Formaldehyde solution (35%)
100 .0 ml
Normal saline solution (0.85%)
900.0 ml
Both solutions were mixed and stored in a capped jar.
4. TRYPAN BLUE SOLUTION FOR INTRADERMAL ASSAY: Solution is injected intravenously to determine hyper-permeability of blood vessels. Trypan blue solution (0.1%) Trypan blue
100 mg
Distilled water 100 ml
5. BUFFERS USED FOR SUSPENDING LIVE CELLS, BACTERIA AND OTHER LIVE MATERIALS Aqueous solution of
Proportion of solutions to prepare buffer Ringer Locke Krebs-Ringer Plain Bicarbonate NaCl, 9 g/lt 100 100 100 100 KCl, 11.5 g/lt 4 4 4 4 CaCl2, 12.2 g/lt 3 3 3 3 KH2PO4, 21.1 g/lt 1 1 MgSO4.7H2O, 38.2 g/lt 1 1 NaHCO3, 13.0 g/lt 3 21 Phosphate buffer, 0.1M, ---
Phosphate 100 4 3 1 20
pH 7.4 Quarter Strength Ringer’s solution NaCl, 2.25 g; KCl, 0.105 g; CaCl2, 0.12 g; NaHCO3 0.05 g dissolve in 1lt water and sterilize by autoclaving (121oC for 15 min) in screw capped vials.
10
Alsever’s Solution Glucose
1.866 g
Sodium chloride
0.418 g
Sodium citrate
0.8
Citric acid
0.055 g
Distilled water to
100
Sodium chloride
8.5 g
g
ml
NSS
Distilled water to make 1 lt.
6. BUFFERS/SOLUTION FOR PLASMID/CHROMOSOMAL DNA ISOLATION 1 M Tris (pH 8.0) Tris base
12.10 g
Distilled water
80.0
ml
Adjusted pH 8.0 with concentrated HCl, make final volume to 100 ml with distilled water. 0.5 M EDTA Disodium ethylene diamine tetraacetate, 2H2O
18.61 g
Distilled water
80.0
ml
Adjust the pH 8.0 by adding Sodium hydroxide pellets (about 2 gm), EDTA dissolve only when pH reaches 8, make final volume to 100 ml with distilled water. Sterilize by autoclaving. 10% SDS Sodium dodecyle sulphate
10.0
g
Distilled water to
100.0 ml
5M NaOH Sodium hydroxide
20.0
g
Distilled water to
100.0 ml
5M Potassium Acetate Potassium acetate
49.075 g
Distilled water to
100.0 ml
3M Sodium Acetate (pH 5.2) Sodium acetate, 3H2O
40.81 g
Distilled water
80.0
ml
Adjust pH 5.2 with glacial acetic acid, make final volume in 100 ml.
11
5M NaCl Sodium chloride
29.20 g
Distilled water to
100.0 ml
10% Cetyl Trimethyl Ammonium Bromide (C-TAB) CTAB
10
g
Distilled water to
100.0 ml
Proteinase K Stock solution of 20 mg/ml in Tris (pH 8, 50 mM) calcium acetate (1.5 mM) solution or in distilled water is prepared aliquoted and kept at –20ºC.
RNase A (free from DNase) Stock solution of 10 mg/ml in Tris chloride buffer (10mM Tris – HCl, pH 7.5 and 15mM NaCl) or 2 mg in 2 ml of TE (100 mM Tris Cl, pH 7.6 and 10 mM EDTA), stored at –20ºC.
7. SOLUTIONS FOR PLASMID ISOLATION BY ALKALINE LYSIS Solution A (5 ml) - Can be stored at 4ºC for 48 hr. Sterile distilled water
4.525 ml
40% dextrose
0.25
1M Tris (pH 8)
0.125 ml
0.5M EDTA
0.1
ml
Lysozyme
25
mg
Sterile distilled water
4.3
ml
10% SDS
0.5
ml
5M NaOH
0.2
ml
Sterile distilled water
2.85
ml
5M Potassium acetate
6.0
ml
Glacial acetic acid
1.15
ml
ml
Solution B (5 ml) – Make Fresh
Solution C (10 ml) - Can be stored at room temperature
TBE Buffer (pH 8.3) 5X 54 gm Tris base, 27.5 gm boric acid, 20 ml of 0.5 M EDTA (pH 8.0) TAE Buffer (pH 7.6)
10X
50 X
1X
Tris base
48.4 gm
242 gm
40 mM Tris acetate
Glacial acetic acid
11.4 ml
57.1 ml
1 mM EDTA
0.5M EDTA (pH 8)
20 ml
100 ml
12
Distilled water to
1000 ml
1000ml
TPE Buffer (1X= 90 mM Tris Phosphate with 2mM EDTA) For 10 × solution 180 gm Tris base, 15.5 ml phosphoric acid (85%, 1.679 gm/ml) and 40 ml of 0.5 M EDTA (pH 8), in 1 lt DW TE Buffer (pH 8) 1M Tris HCl (pH 8)
1.0
ml
0.5M EDTA (pH 8)
0.2
ml
Distilled water to
98.8
ml
Tris Glycine buffer (1X= 25 mM TRis –HCl, 250 mM glycine, 0.1 % SDS) 5 × Solution is made by dissolving 15.1 gm Tris base, 9.4 gm Glycine and 50 ml of 10 % SDS in in 1 lt DW. 50× Ethidium Bromide 10 mg of ethidium bromide is dissolved in 1 ml of distilled water and stored at dark at 4ºC. 6× Gel Loading Buffer Bromophenol blue
0.25%
Xylene cyanol 0.25% Glycerol (v/v) 30% Store at 4ºC.
8. BUFFERS/SOLUTIONS FOR SDS-PAGE Acrylamide-bisacrylamide stock Solution (30% w/v) Acrylamide
29
g
N,N’ methylenebisacrylamide Distilled water to
100
1.0
g
ml
3.0 M Tris HCl (pH 8.8) resolving buffer stock Tris base
36.3
Distilled water 80
g ml
Adjust pH 8.8 with 1 M HCl, (48 ml of 1 M HCl) make final volume to 100 ml. 0.5 M Tris HCl (pH 6.8) Stacking gel buffer stock Tris base
6.0
Distilled water 40
g ml
Adjust pH 6.8 with 1 M HCl, (48 ml of 1 M HCl) make final volume to 100 ml. 0.25 M Tris (pH 8.3) Reservoir buffer stock (10X) Tris
30.3 gm
13
Glycine SDS
144 gm 10 gm
Dist water to make it 1 lt 1.50% APS Ammonium per sulfate
0.15
Distilled water to
ml
10
g
Make fresh after 2-3 weeks if stored at 4oC. 2% SDS Loading Buffer Tris HCl
100
mM
SDS
4
%
Glycerol
20
%
2 Mercaptoethanol
8
%
Bromophenol blue
0.2
%
9. RECIPES FOR MAKING DISCONTINUOUS DISSOCIATING GELS Stock solution
Stacking
Final acrylamide concentration in resolving gel
gel
(%) 20.0
17.5
15.0
12.5
10.0
7.5
5.0
Acrylamide-bisacrylamide
2.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
Stacking gel buffer stock
5.0
----
---
----
-----
-----
----
----
Resolving Gel buffer stock
--
3.75
3.75
3.75
3.75
3.75
3.75
3.75
10 % SDS
0.2
0.3
0.3
0.3
0.3
0.3
0.3
0.3
1.5 % APS
1.0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
Water
11.3
4.45
6.95
9.45
11.95
14.45
16.95
19.95
TEMED
0.015
0.015
0.015
0.015
0.015
0.015
0.015
0.015
10. RECIPES FOR MAKING DISCONTINUOUS NON-DISSOCIATING GELS SDS is not added at any of the stage of preparation of buffers or gels, and Volume is met by DW.
11. RECIPES FOR MAKING CONTINUOUS NON-DISSOCIATING GELS: Same as in number 10 but stacking gel is not used.
14
12. GEL PROCESSING SOLUTIONS: Fixing Solution Methanol 45 % Glacial acetic acid 10 % Distilled water 45 % Staining Solution: It is 0.1% Coomassie brilliant blue R-250 in fixing solution. (0.2 µg to 0.5 µg of any protein in form of sharp band cab be determined with this stain), Silver stain is 100 times more sensitive than it detecting 0.38 ng of protein / mm 2. De-staining Solution Methanol Glacial acetic acid Distilled water Preservative Solution
30 10 60
% % %
Glacial acetic acid Distilled water
7 93
% %
13. ELISA REAGENTS AND BUFFERS A. Coating Buffer Tris buffer 0.1 M, pH 7.0 to 9.0 Or 0.05 M carbonate buffer, pH 9.6 with 0.02 % NaN3 (Sodium azide) or 0.01 M PBS pH 7.4 with 0.02 % NaN3 (Sodium azide) For better coating of antibodies and protein antigens: Dilute serum with equal volume of 0.1 M glycine-HCl buffer, pH 2.5 with 0.1 M NaCl, incubate for 10 min, and neutralize with 0.1 M Tris pH 9.0 Or Make antigen in 0.05 M glycine-HCl buffer, pH 2.5 with 0.1 M NaCl, incubate for 10 min, and neutralize with 0.05 M Tris pH 9.0 For lipid/ surface antigens, make suspension of antigen in sodium deoxycholate (1 mg/ml) solution before coating. b. Washing buffer Phosphate buffered saline (pH 7.2)
1000 ml
Tween-20 (Polysorbate)
0.5 ml
Sodium azide
0.2 gm
Stored at 4°C.
15
C. Blocking solution Gelatin
1.0 g
PBS
100 ml
Or 2 % Bovine serum albumen in water with 0.02 % NaN3 (Sodium azide) or 5% Skim Milk in water with 0.02 % NaN3 (Sodium azide) D. Substrates for Horse radish peroxidase (HRPO) conjugate: 1. Ortho-phenylene-diamine (0.5 mg/ml) in Citrate-phosphate buffer, 0.1 M, pH 4.6 - 5.0), made fresh and add H2O2 (of 30 %, 5 µl per 30 ml) just before use, avoid exposure to direct light. Read at 492 nm. Buffer Stock Solution A Citric acid (C6H8O7.H2O) 2.10 g in Distilled water
100 ml
Buffer Stock Solution B Sodium citrate (Na3C6H5O7.2H2O)
2.94 g in Distilled water
100 ml
Working Solution: 25.5 ml of solution A and 24.5 ml of solution B were mixed just before use. Enzyme activity arrester (1 M, H2SO4) Concentrated sulphuric acid
27.77 ml
Distilled water
72.33 ml
B. Tetramethyl benzidine (TMB): Dissolve 10 mg TMB in 1 ml DMSO and then dilute with 0.1 M sodium acetate citric acid buffer pH 6.0, add 30 µl H2O2 (30 %), read at 450 nm, bright yellow colour after adding sulfuric acid. Enzyme activity arrester (2 M, H2SO4) 3. 5-aminosalycilic acid Add 100 mg in 100 ml of 0.01 M Sod. Phosphate buffer, pH 6.0 having 1mM EDTA, add 20 µl H2O2 (30 %), read at 450 nm, brown colour after adding NAOH. Enzyme activity arrester (1 M, NaOH) e. Substrate for Alkaline phosphatase conjugate 1. p-Nitrophenylene phosphate: Substrate solution (1 mg/ml) is made in 0.05 M carbonate buffer pH 9.8 with 0.001 M MgCl2. Read at 400 nm. Enzyme activity arrester (1 M, NaOH) Or p-Nitrophenylene phosphate solution (1 mg/ml) can be made in 0.05 M diethanolamine buffer pH 9.8. Read yellow colour at 405 nm.
16
Diethanolamine buffer: Diethanolamine, 9.7 ml; Water,
80 ml; Sodium azide 0.02 gm. Adjust
pH with 1M HCl to 9.8. Final volume is made with distilled water to 100 ml. Enzyme activity arrester (3 M, NaOH)
14. SOLUTIONS FOR ESTIMATION OF PROTEIN A. Modified Lowry’s Method (for 0.1 to 1 mg / ml protein) Solution A:1% CuSo4.5H2O
1 g/ 100 ml distilled water
Solution B: 2% sodium potassium tartarate
1 g/ 50 ml distilled water
Solution C: 0.2 M, NaOH
0.8 g/100 ml
Solution D: 4% Na2CO3
4 g/100 ml
Reagent Mixtures Solution E: 49 ml each of solution C and solution D and 1 ml each of solution A and solution B were mixed prior to use. Standard solutions 1% bovine albumin
0.1 g in 10 ml distilled water.
Folin Reagent: To 10 ml Folin Ciocalteau, add 10 ml Distilled water and use it as reagent F. Procedure: To 0.5 ml sample containing up to 0.5 mg protein, mix 2.5 ml of reagent E and keep for 10 min then add 0.25 ml of reagent F and incubate for 30 min at RT. Measure absorbance at 750 nm B. UV Spectrophotometery (0.05 to 2 mg protein ml-1) : Estimation must be made in quartz tubes. Protein in mg ml -1= 1.55 A280- 0.76 A260 Or For increased sensitivity use far UV method (0.01 to 0.05 mg/ml) Protein in mg ml-1= 27 + 120 (A280/ A205) C. Biurate method: (1 to 6 mg/ ml of protein) Biurate reagent: Dissolve 1.5 gm copper sulphate CuSo4.5H2O and 6 gm of sodium potassium tartarate in 500 ml water. Add 300 ml of 105 sodium hydroxide and make up to 1 lt. 1 gm iodine can be added for long term storage of reagent. Procedure: 0.5 ml sample is mixed with 2.5 ml of reagent and allow to stand for 30 min and then read at 540 nm.
17
D. Bicinchoninic acid method: (0.5 to 10 µg/ ml of protein) Solution A: 1.5 % sodium tartarate, 1.6 % NaOH, 8 % Na2CO3 solution is made and pH is adjusted to 11.25 with solid NaHCO3 Solution B: 4 % (w/v) Sodium Bicinchoninic acid Solution C: 4% CuSo4.5H2O Solution D: 4 volumes of C are mixed with 100 volumes of B (made fresh) Solution E: One volume of D 1 volume of A Procedure: To one volume of sample add 1 volume of E, mix and allowed to stand for 60 min at 60 oC. Cool to Rt and read at 562 nm.
E. Dye binding Method: (0.2 to 1.4 mg/ ml of protein or 5-100 µg/ ml in micro assay) Reagent: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of ethanol by vigorous homogenization. Mix it with 100 ml of 85% (W/V) phosphoric acid and then dilute it to 1 lt with DW. Stable at RT for 2 weeks. Procedure: Add 5 ml of the reagent in to 0.1 ml protein sample containing 20-140 µg of protein, mix and allow to stand for 5- 30 min. Measure absorbance at 595 nm. Or For micro assay add 0.8 ml of dye reagent to 0.2 ml of sample containing 1-20 µg of protein, mix and allowed to stand for 5- 30 min. Measure absorbance at 595 nm.
F. Silver binding Method: For highly purified samples and tween-20 should be added to sample for inhibiting adsorption of complexes to glass or plastic surfaces. Reagents: A-7.5 % Tween-20 in 100 mM Tris, 100 mM sodium carbonate. B- 2.5 % Glutaraldehyde (made fresh from stock 25 % solution stored at 4 oC). C- Add 1.4 ml of 20 % w/v sodium hydroxide and 0.2 ml of ammonium hydroxide (29%, concentrated) to 18.2 ml of DW, then add drop wise 0.2 ml of 20 % (w/v) silver nitrate D- 3 % sodium thiosulphate (made fresh). Procedure: Add 11 µl of reagent A to 100 µl of sample containing protein from 15 ng to 2 µg protein. Mix it and centrifuge at 450 g (1000 rpm) for 5 min through a 2 ml Bio-Gel P-2 preequilibrated in 1:10 diluted A and then drain of void volume. Add 0.9 ml of DW to make volume to 1 ml. Then add 20 µl of reagent B and vortex for 2-4 seconds. Then add 200 µl of Reagent C and vortex for 2-4 seconds. Allow to stand for 10 min at RT. Add 40 µl of reagent D and read the absorbance at 420 nm against blank made similarly with 100 µl sample buffer.
18
15. pH OF STANDARD SOLUTIONS OF COMMON CHEMICALS USED IN LABORATORY: S.No. Solution
pH values at 1N
0.1 N
0.01 N
0.001 N
1
Acetic acid
2.4
2.9
3.4
3.9
2
Hydrochloric acid
0.1
1.07
2.02
3.09
3
Sulfuric acid
0.3
1.2
2.1
--
4
Citric acid
--
2.1
2.6
--
5
Ammonium hydroxide
11.8
11.3
10.8
10.3
6
Sodium hydroxide
14
13.7
12.12
11.13
7
Sodium bicarbonate
--
8.4
--
--
8
Sodium carbonate
--
11.5
11
--
16. PHYSICAL CHARACTERISTICS OF COMMON ACIDS AND ALKALIES USED IN LABORATORY S. No.
Substance
Specific gravity
Percent by weight
Norm ality
Ml/lt to make 1M
Eq. Wt.
Mol. Wt.
Mol es/lt
Gms/lt
1
Ammonium hydroxide (stronger Sodium hydroxde (saturated)
0.898
28.0
14.8
67.6
35.0
35.0
14.8
251
1.53 1.11 1.84 1.42 1.40 1.37 1.18 1.05
50.0 10.0 96.0 71.0 67.0 61.0 36.0 10
19.1 2.75 35.9 15.99 14.0 13.3 11.6 2.9
52.4 363.6 55.6 62.5 67.1 75.2 86.2 344.8
40.0
40.0
98.1 63.02
49.0 63.02
19.1 2.75 18.0 15.99 14.0 13.3
36.46
36.46
763 111 1766 1008 938 837 424 105
(syrupy)
1.71
85.0
45.0
55.2
80.0
18.1
1445
(glacial)
1.05 1.045
99.5 36
17.4 6.27
57.5 159.5
60.5
60.5
17.4 6.27
1045 376
1.67 1.54
70.0 60.0
9.2
85.8 108.7
100.5
100.5
11.65 9.2
1172 923
1.52 1.09
50.0 10.0
13.5 1.94
74.1 515.5
126.05 56.10
63.03 56.10
13.5 1.94
757 109
106.00
53.00
381.44
190.72 46.02
23.4
1080
2 3 4
Sulfuric acid H2SO4 Nitric acid (concentrated) HNO3
5
Hydrochloric (concentrated) Dilute Phosphoric acid H3PO4 Acetic acid CH3COOH
6 7
acid
8
Perchloric acid HClO3
9 10
Oxalic acid Potassium hydroxide
11
Sodium carbonate (anhydrous) Borax (Na2B4O7.10H2O) Formic acid HCOOH
12 13
1.20
90.0
23.4
42.7
19
17. BUFFERS FOR SPECIFIC PURPOSES a. McIlvaine’s buffer: Solution A: 0.1 M-citric acid ((2.1015 g of citric acid C6H8O7.H2O, in 100 ml water) Solution B: 0.2 M-disodium phosphate (Na2HPO4, 2.8394 g in 100 ml water) To prepare pH 4.0 solution, add 61.45 ml of solution A and 38.55 ml of solution B. To prepare pH 6.0 solution, add 38.65 ml of solution A and 63.15 ml of solution B. b. Phthalate buffer (0.0125 M, pH 5.0): Solution A: 0.05 m-Potassium hydrogen phthalate (C6H4(COOH)COOK, 1.02115 g in 100 ml water) Solution B: 0.05 M sodium hydroxide (NaOH, 200 mg in 100 ml water) To prepare pH 5.0 buffer, add 50 ml of solution A and 23.85 ml of solution B, dilute with water to make 200 ml then add 5 ml of 1% ethanolic bromocresol purple.
c. PBS, 0.025 M, pH 6.0 and 6.8 SolutionA: 0.025 M Na2HPO4 (Na2HPO4.12H2O, MW 358.16) Solution B: 0.025 M-KH2PO4 (MW 136.09) To prepare pH 6.0 buffer add 12 ml of A and 88 ml of solution B To prepare pH 6.8 buffer, mix 50 ml of slolution A and 50 ml of solution B. (For use in urease or malonate test add 5 ml of 0.1% ethanolic phenol red to 100 ml buffer.) d. PBS, 0.15 M, pH 7.0 Sodium chloride Potassium chloride Di-sodium hydrogen phosphate Potassium dihydrogen phosphate Distilled water to e. PBS, pH 7.2
8.0 0.2 1.15 0.20 1000
Sodium chloride (NaCl)
8.0 g
Potassium dihydrogen phosphate (KH2PO4)
0.20 g
Disodium hydrogen phosphate (Na2HPO4)
1.16 g
Potassium chloride
0.20 g
Distilled water
1000 ml
g g g g ml
The solution was autoclaved at 121°C, 15 lbs pressure for 30 min and stored at 40° for further use. f. PBS, pH 7.4 Sodium chloride
8.0 g
Potassium chloride
0.20 g
Potassium dihydrogen orthophosphate
0.20 g
20
Disodium hydrogen phosphate
1.16 g
Distilled water
1000 ml
g. PBS, pH 7.3, Oxoid tabs) Sodium chloride
8.0 g
Dipotassium hydrogen phosphate
1.21 g
Potassium dihydrogen orthophosphate
0.34 g
Distilled water
1000 ml
h. Azide saline (pH 7.3, Oxoid tabs). Azide act as preservative preventing microbial degradation. Sodium chloride
8.0 g
Dipotassium hydrogen phosphate
1.21 g
Potassium dihydrogen orthophosphate
0.34 g
Sodium azide
0.8 g
Distilled water
1000 ml
i. Borate Calcium saline (pH 7.3). It is used for haemagglutination experiments. NaCl 8g, CaCl2 1 g, H3BO3 1.2 g, sodium borate Deca hydrate (Na2B4O7.10H2O) 0.052 g in 1 lt-distilled water.
j. Veronal-NaCl diluent 5X (used in CFT): Dissolve 5.75 g barbitone in 500 ml hot distilled water, add 85 g of NaCl and make up to 1400 ml. Dissolve 2 g sodium barbitone in 500 ml distilled water and add NaCl-barbitone solution to make upto 2 lt Add 1.68 g MgCl 2.6H2O and 0.28 g CaCl2. For use dilute in 1 in 5 with distilled water. k. Sorensen’s citrate buffer Solution A: 0.1 M-citric acid (2.1015 gm of citric acid C6H8O7.H2O, in 100 ml water) Solution B: 0.1 M-trisodium citrate (Na3C6H5O7.2H2O 2.9411 gm in 100 ml water) To prepare buffer of pH 5.6 mix 13.7 ml of solution A and 36.3 ml of Solution B and adjust the volume to 500 ml with water.
l. Citrate Phosphate buffer for pH 2.6 to 7.0 (0.25 M) Stock Solution A: 1M solution of citric acid (192.1 gm/lt) Stock solution B: 1 M Disodium hydrohen phosphate (Na 2HPO4. 7H2O 536.5 gm/ lt or Na2HPO4. 10H2O 717.1 gm/ lt)
21
A in ml + B in ml to make it 50 ml (i.e. 50 ml-A in ml), dilute to make 100 ml pH
A
pH
A
pH
A
pH
A
pH
A
2.6
44.6
3.6
33.9
4.6
26.7
5.6
21.0
6.6
13.6
2.8
42.2
3.8
32.3
4.8
25.2
5.8
19.7
6.8
9.1
3.0
39.8
4.0
30.7
5.0
24.3
6.0
17.9
7.0
6.5
3.2
37.7
4.2
29.4
5.2
23.3
6.2
16.9
3.4
35.9
4.4
27.8
5.4
22.2
6.4
15.4
m. Citrate Buffer pH 3.0 to 6.2 (0.25 M) Stock Solution A: 1M solution of citric acid (192.1 gm/lt) Stock solution B: 1 M Sodium Citrate (C6H5O7Na3. 2H2O 294.1 gm/ lt) A in ml + B in ml to make it 50 ml (i.e. 50 ml-A in ml), dilute to make 100 ml pH
A
pH
A
PH
A
pH
A
pH
A
pH
A
3.0
46.5
3.6
37.0
4.2
31.5
4.8
23.0
5.4
16.0
6.0
9.5
3.2
43.7
3.8
53.0
4.4
28.0
5.0
20.5
5.6
13.7
6.2
7.2
3.4
40.0
4.0
33.0
4.6
25.5
5.2
18.0
5.8
11.8
n. Phosphate buffer for pH 5.7 to 8.0 (0.25 M) Stock Solution A: 1 M Disodium hydrohen phosphate (Na 2HPO4. 7H2O 536.5 gm/ lt or Na2HPO4. 10H2O 717.1 gm/ lt or Na2HPO4. 2H2O 178.05 gm/ lt ) Stock solution B: 1 M Sodium dihydrohen phosphate (NaH2PO4. H2O 138.35 gm/ lt or NaH2PO4. 2H2O 156.05 gm/ lt ) A in ml + B in ml to make it 100ml (i.e. 100 ml-A in ml), dilute to make 200 ml pH
A
pH
A
pH
A
pH
A
pH
A
pH
A
5.7
6.5
6.1
15.0
6.5
31.5
6.9
55.0
7.3
77.0
7.7
90.5
5.8
8.0
6.2
18.5
6.6
37.5
7.0
61.0
7.4
81.0
7.8
91.5
5.9
10.0
6.3
22.5
6.7
43.5
7.1
67.0
7.5
84.0
7.9
93.0
6.0
12.3
6.4
26.5
6.8
49.0
7.2
72.0
7.6
87.0
8.0
94.7
o. Phosphate buffer for pH 5.8 to 8.0 (0. 5 M) Stock Solution A: 1 M Sodium hydroxide (NaOH 40.0 gm/ lt) Stock solution B: 1 M Potassium dihydrohen phosphate (KH2PO4. 136.00 gm/ lt)
22
A (NaOH) in ml + in 50 ml of B, dilute to make 100 ml pH
A
--
pH
A
pH
A
pH
A
pH
A
pH
A
6.1
6.8
6.5
13.9
6.9
25.9
7.3
37.0
7.7
43.5
5.8
3.6
6.2
8.1
6.6
16.4
7.0
29.1
7.4
39.1
7.8
44.5
5.9
4.6
6.3
9.7
6.7
19.3
7.1
32.1
7.5
40.9
7.9
45.3
6.0
5.6
6.4
11.6
6.8
22.4
7.2
34.7
7.6
42.4
8.0
46.1
p. Potassium Phosphate buffer for pH 5.8 to 8.0 (0. 1 M) A: Volume of 1 M K2HPO4 (ml) to make 100 ml with 1 M KH2PO4 and then finally dilute with distilled water to make 1 lt. pH
A
pH
A
pH
A
pH
A
pH
A
pH
A
5.8
8.5
6.2
19.2
6.6
38.1
7.0
61.5
7.4
80.2
7.8
90.8
6.0
13.2
6.4
27.8
6.8
49.7
7.2
71.7
7.6
86.6
8.0
94
q. PBS (Bacteriological) for 1 lt. 8 gm (137 mM) NaCl, 0.2 gm (2.7 mM) KCl, 1.44 gm (10 mM) Na2HPO4,
0.2 gm (2
mM) KH2PO4 Dissolve in 800 ml Dist. Water, adjust pH 7.4 with HCl and make it to 1 lt. If required it may be supplemented with CaCl2, 1 mM and MgCl2 0.5 mM
r. Tris Buffer To make 0.05 M Tris buffere ad 0.1N HCl to desired level in 0.1 M Tris base 50 ml and finally dilute to make 100 ml with Distilled water. (More the concentration more the pH, 10mM and 100mM buffers differ by 0.1 pH unit) pH
HCl
pH
HCl
pH
HCl
pH
HCl
pH
HCl
pH
HCl
7.1
45.7
7.4
42.0
7.7
36.6
8.0
29.2
8.3
19.9
8.6
12.4
7.2
44.7
7.5
40.3
7.8
34.5
8.1
26.2
8.4
17.2
8.7
10.3
7.3
43.4
7.6
38.5
7.9
32.0
8.2
22.9
8.5
14.7
8.8
8.5
To make 1 M Tris buffer: 121.1 gm Tris base, dissolve in 800 ml distilled water add required quantity of concentrated HCl and make it a lt with DW. pH
HCl
pH
HCl
pH
HCl
7.4
70 ml
7.6
60 ml
8.0
42 ml
23
s. Barbital Buffer (0.05 M) pH 6.8 to 9.2 Stock Solution A: 0.2 M solution of sodium barbital (41.2 gm/lt) Stock solution B: 0.2 M HCl In 50 ml of A add desired volume of B and finally dilute to make 200 ml pH
B
pH
B
pH
B
pH
B
pH
B
6.8
45.0
7.4
32.5
8.0
17.5
8.6
6.0
9.2
1.5
7.0
43.0
7.6
27.5
8.2
12.7
8.8
4.0
7.2
39.0
7.8
22.5
8.4
9.0
9.0
2.5
t. Borate Buffer (0.0125 M) pH 8.1 to 9.0 Stock Solution A: 0.025 M solution of sodium borate Na2B4O7. 10H2O (9.525 gm/lt) Stock solution B: 0.1 M HCl In 50 ml of A add desired volume of B and finally dilute to make 100 ml pH
B
pH
B
pH
B
pH
B
pH
B
8.1
19.7
8.3
17.7
8.5
15.2
8.7
11.6
8.9
7.1
8.2
18.8
8.4
16.8
8.6
13.5
8.8
9.4
9.0
4.6
u. Borate Buffer (0.0125 M) pH 9.3 to 10.7 Stock Solution A: 0.025 M solution of sodium borate Na2B4O7. 10H2O (9.525 gm/lt) Stock solution B: 0.1 M NaOH In 50 ml of A add desired volume of B and finally dilute to make 100 ml pH
B
pH
B
pH
B
pH
B
pH
B
9.3
3.6
9.6
11.1
9.9
16.6
10.2
20.5
10.5
22.7
9.4
6.2
9.7
13.1
10.0
18.3
10.3
21.3
10.6
23.3
9.5
8.8
9.8
15.0
10.1
19.5
10.4
22.1
10.7
23.8
v. Glycine Buffer (0.05 M) pH 8.6 to 10.6 Stock Solution A: 0.2 M solution glycine (15.1 gm/lt) Stock solution B: 0.2 M NaOH In 25 ml of A add desired volume of B and finally dilute to make 100 ml pH
B
pH
B
pH
B
pH
B
pH
B
8.6
2.0
9.0
4.4
9.4
8.4
9.8
13.6
10.4
19.6
8.8
3.0
9.2
6.0
9.6
11.2
10.0
16.0
10.6
22.75
24
w. Sodium Carbonate bicarbonate Buffer (0.05 M) pH 9.2 to 10.8 Stock Solution A: 0.1 M solution of sodium carbonate, Na2CO3. 10H2O (28.62 gm/lt) Stock solution B: 0.1 M solution of sodium bicarbonate, NaHCO3 (8.40 gm/lt) Add desired volume of B and make it to final volume of 100 ml with solution A pH
at pH
pH at 20oC
at B
pH
at B
pH at 20oC
37oC
pH
at B
20oC
37oC
37oC
9.2
8.8
90
9.8
9.5
60
10.3
10.1
30
9.4
9.1
80
9.9
9.7
50
10.5
10.3
20
9.5
9.4
70
10.1
9.9
40
10.8
10.6
10
x. Carbonate Buffer (0.025 M) pH 9.7 to 10.9 Stock Solution A: 0.05 M solution of sodium bicarbonate, NaHCO3 (4.20 gm/lt) Stock solution B: 0.1 M NaOH Add desired volume of B and make it to final volume of 100 ml with solution A pH
B
pH
B
pH
B
pH
B
pH
B
9.7
6.2
10.0
10.7
10.3
15.2
10.6
19.1
10.9
22.0
9.8
7.6
10.1
12.2
10.4
16.5
10.7
20.2
9.9
9.1
10.2
13.8
10.5
17.8
10.8
21.2
18. ANTIBIOTIC SOLUTIONS (must be stored at -20oc in aliquots) Antibiotic
Amoxicillin Amphotericin B Ampicillin Aztreonam Carbenicillin Cefotaxime Ceftazidime Cephelothins & Cephalosporins Chloramphenicol Cinoxacin Clavulanic acid Clindamycin HCl Enoxacin Erythromycin Gentamicin Kanamycin
Conc. mg/m l 50 5 50
Dissolved in
Sterilized through
Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter
30 50 25 30 30
PBS, 0.1 M, pH 6 Dimethylformamide Distilled water, PBS 0.1 M pH 8 Aqueous NaHCO3 Distilled water Dimethyl Sulfoxide PBS, 0.1 M, pH7.0 PBS, 0.1 M, pH 6.0
34 20 10 50, 10 10 25 50 10
Ethanol/ methanol NaOH, 0.1 M Distilled Water Distilled Water, Methanol NaOH, 0.1 M Distilled Water Distilled water Distilled water
Not needed Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter
Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter Filtration 0.22 µm filter
25
Moxalactum 30/ 50 HCl, 0.04 – 0.08 M Filtration 0.22 µm filter Nalidixic acid 30/ 50 NaOH, 0.1 M Filtration 0.22 µm filter Norfloxacin 30 NaOH, 0.1 M Filtration 0.22 µm filter Ofloxacin 30 NaOH, 0.1 M Filtration 0.22 µm filter Oxolinic acid 10 NaOH, 0.1 M Filtration 0.22 µm filter Polymyxin B sulphate 10 PBS, 0.1 M, pH-7.0 Filtration 0.22 µm filter Rifampin 30 Methanol/ DMSO Not needed Streptomycin 10 Distilled water Filtration 0.22 µm filter Sublactum-sodium salt 50 Distilled Water Filtration 0.22 µm filter Sulfonamides 200 NaOH, 0.1 M Filtration 0.22 µm filter Tetracycline 50 Ethanol Not needed Trimethoprim 30/ 50 HCl, 0.05 M Filtration 0.22 µm filter ++ Mg are antagonistic to tetracycline therefore, use media lacking Mg++ while using tetracycline for selection.
19. SUGAR SOLUTIONS (concentration in % w/v) (can be stored at -20oc in aliquots) Class
Sugar
Concentration
Class
Sugar
Concentration
Pentoses
Arabinose
40
Polysachharides
Glycogen
5
Xylose
50
Inulin
20
Methyl
Rhamnose/
40
Starch
Soluble in hot
pentose
isodulcitol
Hexoses
Fructose
50
Galactose
Disachharide
Trisaccharide
water only Glycosides
Aesculin
0.1, 7.5 in hot
30
Amygdalin
7.5
Glucose/dextrose
50
Arbutin
10
Mannose
50
Salicin
3
Sorbose
40
Adonitol
40
Cellobiose
10
Dulcitol
5, on heating
Lactose
15
Erythritol
40
Maltose
50
Glycerol
Any proportion
Melibiose
10
Mannitol
15
Sucrose
50
Sorbitol
50
Trehalose
25
Inositol
15
Melezitose
10
Raffinose
10
Alcohols
Non-carbohydrate
Sterilized either through filtration, tydallization or a few by autoclaving for 10 min at 110 oC.
26
20. DYE SOLUTIONS AND INDICATORS a. Common dyes Indicator
Concentration ml of 0.05 N in solution
Solvent
pH Range
NaOH/g
Colour Acid
Alkaline
indicator Methyl red
0.2
0
50% ethanol
4.2-6.3
Red
Yellow
Chlorophenol red
0.2
47
50% ethanol
4.8-6.4
Yellow
Purple
Andrade’s (Acid
0.5
0.15-0.18 N
Water
5-8
Pink
Yellow
40% ethanol
5-8
Red
Blue
50% ethanol
5.2- 6.8
Yellow
Purple
6.0- 7.6
Yellow
Blue
50% ethanol
6.8- 8.0
Red
Yellow
Fuchsin)
sol. is used
Litmus
25
Bromocresol
0.2
1N HCl needed 37
purple Bromothymol
or water 0.2
32
blue Neutral red
0.1
Phenol red
0.2
57
50% ethanol
6.8- 8.4
Yellow
Red
Cresol red
0.2
53
or water
7.2- 8.8
Yellow
Red
Thymol Blue
0.2
43
1.2- 2.8
Red
Yellow
8.0- 9.6
Yellow
Blue
Phenolphthalein
0.1
0
50% ethanol
8.3- 10.0
Colorless Red
Congo Red
0.1
0
Water
3.0- 5.2
Blue
Red
Note: all dyes are first dissolved in small volume of solvent in a grinder. b.
Andrade’s Indicator: Dissolve 0.5 g Acid Fuchsin in 100 ml distilled water, add 1N NaOH till the colour of solution is straw yellow (usually 15-18 ml). Keep on RT for a day and again check the colour and if needed add more NaOH solution (1ml) to have the straw yellow colour. The solution is used at the rate of 1% in peptone water (at this concentration it should not raise pH of PW above 7.2 at which the indicator is yellow in colour.
c. Litmus solution: 2.5 g of litmus granules are ground into 50 ml of 40% ethanol, boil for a minute and filter through Whatman filter paper No. 1 or just take the supernatant by decanting and add 50 ml of 40% ethanol, boil for a min. Centrifuge at 5000 g and take supernatant adjust volume with 40% ethanol to 100 ml and add 1N HCl drop by drop until solution is purple. Use the indicator solution @ 2.5% in medium.
27
21. COMMON CULTURE MEDIA USED IN MICROBIOLOGY A. Tryptic soy broth Bacto tryptone
17.00 g
Bacto soytone
3.00 g
Sodium chloride
5.00 g
Dipotassium phosphate
2.50 g
27.5 g rams of the medium was dissolved in 1 litre of distilled water and sterilized at 121°C for 15 min. The pH was adjusted to 7.30.2.
B. Semisolid Phosphate Buffered Agar (pH 7.4) Meat extract
5.0
g
Peptone
10.0
g
Sodium chloride
3.0
g
Di-sodium hydrogen phosphate
2.0
g
Agar
10.0
g
Distilled water to
1000
ml
Beef extract
3.0
g
Peptone
5.0
g
Gelatin
120.0 g
Distilled water to
1000
ml
Bacto peptone
10
gm
Bromophenol blue (0.2%)
12
ml
Distilled water to
1000
ml
C. Gelatin Agar
D. Organic Acid Medium
Bromophenol blue is added after adding sugar @1% and adjusting the pH 7.4. E. Decarboxylase Test Media Bacto peptone
5.0
g
Yeast extract
3.0
g
Glucose
1.0
g
Bromocresol purple (1.6%)
1
ml
Distilled water to
1000
ml
Bromocresol purple is added after adding the amino acid @1% and adjusting pH to 6.8.
28
F. Luria Bertani Broth (pH 7.4) Tryptone
10.0
g
Yeast extract
5.0
g
Sodium chloride
10.0
g
Distilled water to
1000
ml
Bacto peptone
10.0
g
Sodium chloride
5.0
g
Andrader indicator
10
ml
Distilled water to
1000
ml
G. 1% Peptone Water (pH 7.4)
H. RPMI-1640 growth medium RPMI-1640
10.40 g
HEPES (SRL, India)
5.92 g
ß-mercaptoethanol
100.00 µl
Sodium bicarbonate (NaHCO3)
2.20 g
L-glutamate
2 mM
Foetal calf serum
100 ml
Streptomycin
100 µl/ml
Penicillin
100 IU/ml
Tripple distilled water made up to
1000 ml
The prepared RPMI-1640 media was sterilized bymembrane filteration (0.22 µm), sterility was tested for 48 h and stored at 4°C for further use. I. MEM (minimum Essential Media) [(Eagles’) Glassgow modification, M/S Microlab, Mumbai) MEM
10.60 g
Penicillin
1 lakh units
Streptomycin
1.0 g
Tryptase phosphate broth (Difco)
5.0 g
NaHCO3
1.0 g
L-glutamine (Sigma)
300 mg
Distilled water
1000 ml
29
J. M-9 agar (Zyskind J. W. and Berstein, S. I. (1989) Disodium hydrogen phosphate (Na2HPO4)
6.00 g
Potassium dihydrogen phosphate (KH2PO4)
3.00 g/litre
Sodium chloride (NaCl)
5.00 g/litre
Ammonium chloride (NH4Cl)
1.00 g/litre
Agar
10 g/litre
K. aro mix (Coulson et al., 1994) p-aminobenzoic acid
10.00 µl/ml
Dihydroxybenzoic acid
10 µg/ml
Phenylalanine
40 µg/ml
Tyrosine
40 µg/ml
Histidine
40 µg/ml
Tryptophan
40 µg/ml
22. CLINICAL SAMPLE TRANSPORT MEDIA Components
Sodium Chloride Potassium Chloride Calcium Chloride Magnesium Chloride Mono potassium Phosphate Disodium phosphate Sodium Thioglycollate Charcoal Sodium Glycerophosphate Methylene blue Agar pH
Amount in grams per litre Amies Transport
Amies Transport
Stuart Transport
Carry-Blair
medium (with
medium (without
medium
transport medium
charcoal)
charcoal)
3 0.2 0.1 0.1 0.2
3 0.2 0.1 0.1 0.2
1.15 1
5 0.1
0.1
1.15 1
0.9
1.1 1.5
10 00
00 00
10
4 7.3 (0.2)
4 7.3 (0.2)
0.002 3 7.4 (0.1)
5 8.4 (0.2)
Heat to boil and then dispense in screw capped vials and autoclave at 121OC. Swabs can be inserted in to medium for transport, should be transported as soon as possible within 24 hr to the laboratory for processing.
30
23. PRE-ENRICHMENT MEDIA AND RESUSCITATION MEDIA Ingredients
Peptone Tryptone Monopotassium Phosphate Disodium Phosphate Sodium chloride Dextrose Magnesium sulphate Ferric ammonium citrate Sodium pyruvate pH
Amount in grams per litre Universal pre-
Modified Buffered
enrichment broth
peptone water
5 5 15
10
10
3
1.5
7 5 0.5 0.25 0.1
7 5
3.5 5
7.2 (0.2) For enteropathogens
7.2 (0.2) For enteropathogens.
0.2 6.3 (0.2) Good for all types of microbes
Buffered peptone water
Boil to dissolve, dispense in tubes and autoclave to sterilize at 121 OC.
24. STORAGE OF CULTURES FOR FUTURE USE A. On/in media a. Dorset Egg Medium It is one of the mediums of choice for maintenance of smooth (S) phase of bacteria. It is suitable for storage for 10-12 years if kept well stoppered in screw capped tubes. It is made as per the following composition: Normal saline solution
500 ml
Egg contents (albumin + yolk)
500 ml
This medium is dispensed in medium sized screw capped tubes to fill up to half length and cotton plugged to sterilize in slanted position kept in a water bath for 1 hour at 80°C (temperature is taken as on at top of the cover of water bath) and allowed to cool. Sterilize similarly for 3 consecutive days. Tubes are then incubated to test the sterility and then hermetically sealed by caps. Tubes are surface and stab-inoculated. After overnight incubation, the tubes are labeled and stored in the dark at room temperature or at 4-10OC. Dorset medium can also be sterilized by autoclaving the inspissated egg medium tubs at o
121 C for 15 min. For autoclaving tubes must be tightly capped, otherwise steam bubbles may break the medium.
31
b. Buffered semisolid Nutrient agar Most cultures remain viable for several years when stored in well-stoppered (screw-capped) tubes, in cold and dark. It is made as per the following composition: Meat extract-
5gm
Peptone-
10gm
NaCI-
3gm
Na2HPO4. 12 H2O
2gm
Agar-
10gm
Distilled water
1 ltr
pH
7.4
This medium boiled for 10 minutes to dissolve the agar and other ingredient and is dispensed in small tubes, sterilized for twenty minutes at 110°C and allowed to cool. Small 9 mm × 9 mm tubes (Screw capped or Kahn’s tubes) , hermetically sealed by caps, cork or rubber stoppers as per tube type The tubes are plugged with cotton wool for sterilization and the cotton wool is replaced by sterile stoppers after inoculation. Tubes are stab-inoculated. After overnight incubation, the tubes are carefully labeled, sealed with paraffin or parafilm and stored in the dark at 4 to 8OC or at room temperature. Refrigeration is not necessary at least in temperate climate. When the stored strains need to be used again, a portion of the culture is transferred to a sterile trypticase soy or nutrient broth with an inoculating loop. After overnight incubation, a loopful of broth culture is streaked on nutrient agar. When isolated colonies develop on the agar, a smooth or a rough colony is selected as needed.
c. Glycerol broth It is a liquid medium, which is good for maintaining strains in S form, it contains trypticase soy broth diluted 1/2 with distilled water, 75 ml; Glycerol, 25 ml (pH 7) This medium is distributed in leak-proof small tubes, screw-capped and sterilized by autoclaving for twenty minutes at 110°C. Culture from a nutrient agar is suspended (heavy suspension) directly in the medium. It may be conserved for several years at 10°C. The tubes can also be stored at -70°C or -80°C for long-term storage. d. Stock culture Agar: For maintenance of stock cultures of bacteria specially Streptococci. BHI broth 1lt, Gelatin 10.0g, Casein 5.0g, Dextrose 0.5g, Na 2HPO4 4.0g, Sodium citrate 3.0g, Agar 7.5g, pH 7.5, autoclave in screw-capped tubes. It keeps Streptococci culture in good condition for >4 months at room temperature.
32
Lactobacilli Agar (Difco) can also be used for maintenance of cultures. e. In sterilized powder of Multani Mitti (Fullers earth): Pour 1 ml culture to be preserved in 2ml screw cap tube containing 0.5 g of Multani Mitti (finely grounded). Screw the tube and store at 4oC, most cultures remain viable for a few years.
B. Freezing the cultures Freezing (-70°C or -80°C) is recommended for long-term preservation of cultures. For freezing a thick bacterial suspension in nutrient broth plus 15% (v/v) glycerol is distributed in sterile (10 × 30 mm) screw-capped vials or neutral glass ampoules containing about 30 sterile glass embroidery beads (1-2 mm in diameter) or a sterilized filter paper strip (5mm × 50 mm) and the excess suspension poured off from the vial. Vials are capped and ampoules are fire sealed hermetically, placed in suitable racks and stored at -70°C or -80°C as such. To recover a frozen strain, a vial is removed from the freezer and one bead or paper strip is picked up using a platinum loop or forceps or spatula. The vial is then immediately replaced in the freezer. The removed bead is transferred to a nutrient broth or TSB tube and incubated.
C. Freeze-drying Freeze-drying is done for long-term preservation of cultures. For freeze-drying a thick bacterial suspension in horse serum plus 15% (v/v) glycerol (culture is harvested in medium itself) or in other suitable medium* is distributed in sterile neutral glass ampoules containing a sterilized filter paper strip (5mm × 50 mm). Vials are placed in suitable freeze dryer. Freeze-dried cultures are sealed hermetically when ampoules are still on machine with a acetylene or gas flame. Ampoules are stored after labeling at 0 to 4 OC or at -20°C or -80°C as per available facilities. To recover a frozen strain, an ampoule is removed from the freezer and paper strip is picked up using forceps and is transferred to a nutrient broth or TSB tube and incubated. *Medium for freeze drying of cultures: Medium is made in sterilized glass distilled water and medium is sterilized through filtration only. It contains: Bacto caseitone (2.5%), Sucrose 2.5%) and Sodium glutamate (1% ).
25.
SOME
SPECIAL
MEDIA
AND
REAGENTS
REQUIRED
IN
BACTERIOLOGY a. Media for blood cultures in cases of typhoid: 10 ml of blood should be added to 100 ml medium and incubated at 37oC and plated daily for up to 11 days. Addition of liquoid (sodium
33
polyanethol sulphonate, 0.03-0.05%) or bile in enrichment interferes with bactericidal action of blood and improves the bacterial detection. Liquoid broth (0.05%) Liquoid solution (5% in 0.85% NSS)
10 ml
Nutrient broth
1 lt
pH 7.6. Distribute in tubes, sterilize by autoclaving at 121oC for 15 min. Instead of liquoid one can use citrate-saponin broth culture of bacteria from blood; composition is follows: Sodium citrate (Na3C6H5O7. 2H2O)
2g
Saponin, white (BDH)
1g
Sterilized nutrient broth
1 lt
pH
7.6. o
Never heat above 100 C. Sterilize at 100oC for 20 min on steam for three consecutive days. b. Swarm-agar (Gard plate) Agar concentration of the nutrient agar made for H phase inversion in a very important determinant of success. It may vary from 0.5% to 1 % depending upon the batch of agar used. The agar medium must be sufficiently soft for spot-inoculated motile bacteria to swarm over the medium after overnight incubation at 37°C. Preliminary trials, without addition of antiserum, are therefore necessary before using a batch of medium. Nutrient agar (pH 7.4) prepared for that purpose or ordinary laboratory nutrient agar made semi-solid by the addition of broth in a proportion allowing overnight swarming. The volume required for a 90 mm petri dish is about 30 ml. swarming on the surface is easier if sodium desoxycholate (0.15 to 0.3 g/l) or bile salt (0.5 g/l) is added to the medium. c. Semi-solid agar in U tubes Nutrient broth
l000ml
Agar
2g
Potassium nitrate
1 g (to inhibit gas production)
pH
7.4
Alternatively, for phase inversion, a tube containing the same semi-solid medium and an inner tube open at each end with the upper end extending above the surface of the medium can be used (Craigie). The small inner tube is inoculated and a motile culture will migrate down through the inner tube and then to the surface of the tube outside the inner tube. d. Worfel Ferguson Medium for capsule enhancement Potassium sulphate
1 gm
34
Sodium chloride
2 gm
Magnesium sulphate
0.25 gm
Sucrose
20 gm
Yeast extract
2 gm
Agar
15 gm
Distilled water
1 lt
Autoclave to sterilize.
e. Minimal Salt Medium (Vogel Bonner Medium E (50X) Distilled water at 45oC 670 ml MgSO4. 7H2O
10 gm
Citric acid monohydrate 100 gm K2HPO4 anhydrous
500 gm
Sodium ammonium
175 gm
Phosphate. 4 H2O Allow each salt to dissolve first than add the other one
f. Glucose Minimal Salt Agar Medium 15 gm Agar, 20 ml 50× Minimal Salt Medium, 930 ml Distilled water, 50 ml of 40% glucose g. Minimal Agar Medium 2 gm glucose, 1 gm (NH4)2SO4 7 gm K2HPO4, 0.5 gm MgSO4 15 gm Agar, 1 lt Distilled water h. Deca strength phage broth (DSPB) 100 gm peptone, 25 gm NaCl 50 gm yeast extract 80 gm K2HPO4 in 1 lt. Dist. Water, pH 7.6.
35
26. BACTERIOLOGICAL CULTURE MEDIA USED IN BIOCHEMICAL CHARACTERIZATION OF BACTERIA A. MR-VP test medium (Glucose phosphate medium): Dissolve 5 g, K2HPO4 and 5g peptone in 1000 ml distilled water, steam and adjust pH to 7.5, add 5 g of glucose and then distribute in 1.5-2 ml aliquot in to tubes and then sterilize at 115 oC for 10 min.
B.
Dextran and Levan production medium (Sucrose agar): Melt the 1000 ml of NA or Digest agar (meat extract after digestion with pancreatic juice) and add 50 g of sucrose, steam for 30 min, then aseptically add 50 ml of serum when medium is cooled to 55oC and pour the plates.
C.
Sucrose broth: Dissolve 50 g of sucrose in 1000 ml of infusion broth, tube the medium and sterilize by steaming for 1 h.
D.
Starch Agar: Dissolve 10 g of soluble starch (potato starch) in 50 ml of distilled water, mix it in molten NA and sterilize at 115oC for 10 min and pour the plates.
E.
Aesculin broth: Dissolve 1 g aesculin and 0.5 g of ferric citrate in 1000 ml of PW, sterilize at 115oC for 10 min. Agar can be made by adding 2% agar before sterilization. To make aesculin bile agar, 40 g bile salt (ox bile) is added before sterilization in to Aesculin agar.
F.
Aesculin Blood agar: Make 2% (W/V) aesculin and 1% (W/V) ferric citrate solution together and steam for 5 min at 100oC. On well-dried BA plate pour 1 ml of aesculin-ferric citrate solution and allow drying. To test, spot inoculate the medium and incubate to observe blackening of medium under and around colonies of aesculin hydrolyzing bacteria.
G.
Capsulation medium for Bacillus anthracis (Serum-Bicarbonate agar): Add 0.3 g yeast extract, 0.5 g glucose and 2.5 g agar in 100 ml of NB, sterilize at 115 oC for 10 min, cool to 55oC and add filter sterilized 11 ml each of 7% NaHCO3 and 7% BSA fraction V to have 0.7% final concentration of each, mix and pour the plate. Instead of BSA fraction V, any filter sterilized serum can be added @ 20% (V/V) final concentration.
H.
Casein agar: Add 50 ml of pre-sterilized (at 115oC for 10 min.) skimmed milk in 50 ml of molten and cooled to 55oC double strength NA, mix together and pour the plates. Instead of skim milk one can use 10% solution made with skim milk powder.
36
I.
Christensen’s Citrate medium: Dissolve 3 g sodium citrate, 0.2 g glucose, 0.5 g yeast extract, 0.1 g L-cysteine HCl, 0.4 g ferric ammonium citrate, 1 g KH2PO4, 5 g NaCl, 0.08 g Na2S2O3 and 20 g agar in 1000 ml of water by steaming for 10-15 min, adjust pH to 6.8-6.9 and then add 6 ml of 0.2% aqueous phenol red. Aliquot in suitable 5 ml tubes and sterilize at 115 oC for 20 min, cool in slanted position.
J.
Castaneda medium: It is biphasic medium for isolation of pathogens from blood like Brucellae, it consists of solid and liquid phase. In flat bottle, 30 ml glucose serum agar is allowed to set on one of the narrow side and then 20 ml sterile glucose serum broth is added to each of the bottle. Sealed with rubber cork, incubated for 24 hr to test sterility.
K.
Koser’s Citrate medium: Dissolve 5 g NaCl, 0.2 g MgSO4.7H2O, 1 g NH4H2PO4, 1 g K2HPO4, in 1000 ml water then add 2 g citric acid, adjust pH to 6.8 with N-NaOH, dispense in 5 ml aliquots in screw capped tubes and autoclave at 115 oC for 20 min.
L.
Simmons’ citrate: It is same as Koser’s citrate with added 0.008% bromothymol blue (20 ml of 0.4% aqueous solution/lt) and 2% (W/V) agar. Medium is made in slants.
M. Organic acid medium: To determine ability of bacterium to use an organic acid as carbon source. Dissolve 1 g NaCl, 0.2 g MgSO4.7H2O, 1 g (NH4)2HPO4, 0.5 g K2HPO4, and 2 g of test organic acid in 200 ml water. Dissolve 20 g agar by boiling in 800 ml water. Mix both the solutions and adjust pH to 6.8. Thereafter, add 4 ml of 0.2% aqueous Phenol red. Aliquot in 2-3 ml tubes and autoclave at 115oC for 20 min.
N.
Decarboxylase medium: For determining decarboxylation of lysine, arginine and ornithine amino acid, however other amino acids may be used in the medium. L-amino acids are used @1% but DL-amino acids are used @2% level. To make Moller’s medium (1955), dissolve by heating 5 g peptone, 5 g beef extract, 5 mg pyridoxal, and 0.5 g glucose in 1 lt of water, adjust pH 6.0 and add 5 ml of 0.2% bromothymol blue and 5 ml of 0.2% cresol red aqueous solution. Finally add required amino acid at the required level i.e., 1% for L and 2% for DL amino acids. Always keep some of the medium without amino acids for control. Distribute final medium in 1.5- 2 ml volume in screw-capped tubes and sterilize at 115oC for 10 min. Falkow modified medium (1958) is made by dissolving 5 g peptone, 3 g yeast extract and 1 g glucose in 1 lt water, pH is adjusted at 6.7 and then 10 ml of 0.2% bromocresol purple is added, then required
37
L-amino acids are added @0.5%. Distribute final medium in 1.5- 2 ml volume in screw-capped tubes and sterilize at 115oC for 10 min.
O.
Hippurate hydrolysis medium: Sodium hippurate is added in to NB @ 1% (w/V) level and sterilized in screw-capped tubes at 115oC for 20 min. Filter sterilized 10% Sodium hippurate can be added after sterilization of NB. For Streptococci medium should also contain 0.1% final concentration of glucose, filter sterilized glucose is added after sterilization of the medium.
P.
KCN broth: Dissolve 3 g peptone, 5 g NaCl, 0.225 g KH2PO4, 5.64 g Na2HPO4.2H2O in distilled water by boiling. Distribute in 100 ml volumes and autoclave to sterilize at 115 oC for 20 min. For use add 1.5 ml of 0.5% freshly prepared KCN solution (in sterilized distilled water) in 100 ml of base and distribute in sterile screw-capped tubes.
Q.
Loeffler’s Serum slants: Mix 5 g glucose in 250 ml NB, steam sterilized for 30 min and finally mix it with 750 ml of filtered serum. Distribute in 2.5 ml volumes in suitable tubes and tyndallize (steam at 75oC for 2 h daily for 3 consecutive days) the tubes in slanted position.
R.
LJ medium (Lowenstein-Jensen medium for Mycobacterium): Take and mix contents (albumin and yolk) of 20 medium sized chicken eggs dipped in 70% ethanol for 15 min and then wiped with sterile gauze. Filter the egg contents through sterilized gauze. Mix the contents with 600 ml of autoclaved salt solution (containing 2.4 g KH 2SO4, 0.2 g MgSO4.7H2O and 12 ml glycerol) and 10 ml of 2% malachite green. Distribute in 5 ml volumes in 30 ml screw cap bottles. Screw the caps tightly and place the bottles almost horizontally in hot air oven raise the temperature slowly to 75oC to inspissate and then raise temperature to 80oC and maintain for 45 min.
For characterization of Mycobacterium LJ medium can be made with TSC (thio-semicarbazone: thioacetazone) by adding 1.8 ml TSC (0.6 ml of 0.5% TSC made in dimethyl formamide + 1.2 ml distilled water) in 300 ml of LJ medium before inspissation to give final concentration of 10 mg/ ml. LJ medium is also prepared with PNB (p-nitrobenzoic acid). In 600 ml of LJ medium 300 mg of PNB is added after dissolving it as under. Dissolve 300 mg PNB in 10 ml water containing one drop of 0.4% phenol red and 2 ml of 1 N-NaOH, then add 1N- HCl drop by drop till the solution turns yellow and precipitate appears thereafter, re-titrate with 1 N-NaOH to re-dissolve the precipitate and have just pink colour.
38
S.
Lecithinase agar/ Lecithinovitellin agar: Collect 50 ml egg yolk (from 4 chicken eggs), mix with 1 lt of NSS (0.85% NaCl), mix well filter through sterile gauze and aliquot in 100 ml volumes after filter sterilization (can be stored at 4-8oC for about one month). Add 100 ml of egg yolk solution in 900 ml molten NA (cooled at 55oC) and pour the plates.
Alternate method: Take one egg, dip in 70% ethanol for 15 min, wipe it with sterile gauze, break the egg in sterile plate with all aseptic measures, carefully pipette 5 ml of yolk in to 1 lt of molten NA, mix with same pipette. Pour the plates.
T.
Malonate-phenylalanine medium: Dissolve 2 g (NH4)2SO4, 0.6 g K2HPO4, 0.4 g KH2PO4, 2 g NaCl, 3 g Na-Malonate, 2 g DL-phenylalanine or 1 g L-phenylalanine and 1 g yeast extract in 1 lt distilled water by boiling, add 12.5 ml of 0.2% aqueous Bromothymol blue. Dispense in 2 ml aliquot in screw-capped tubes and sterilize at 115oC for 20 min.
U.
Litmus milk: Refrigerate the whole milk overnight and siphon out the creamy layer, steam for 1 h and again refrigerate, skim the cream and filter, add Litmus solution @2.5% level (V/V), tube in suitable volume and sterilize at 115 oC for 10 min or tyndallize. Overheating causes caramelization of milk and interferes with the bacterial action. Homogenized and market skim milk is unsuitable.
V.
Purple milk: Same as Litmus milk, instead of litmus solution, 10 ml 0.2% Bromocresol purple is added in to 1 lt milk.
W. Purple milk (Ulrich, 1944): Same as litmus milk but indicators are 7.5 ml of 0.2% solution of chlorophenol red (pH indicator) and 2.5 ml of 0.2% methylene blue (redox indicator) in 1 lt of milk before sterilization.
X.
Motility agar: Add 80 g gelatin and 4 g agar in 1 lt Nutrient broth, tube the medium and sterilize at 115oC for 20 min. For simultaneous detection of H2S add 0.2 cysteine, 2 g sodium citrate and 0.2 g ferrous ammonium sulphate is added to hot NB and then add agar and gelatin as above. Citrate acts as clarifying agent.
Y. Sulphide Indole motility agar: Dissolve 30 g tryptone, 3 g meat extract, 0.5 g Na 2S2O3.5H2O, 0.2 g cysteine-HCl, 5 g NaCl in 1 lt water and add 5 g agar, boil to dissolve all ingredients and tube in screw-capped bottles to sterilize at 121oC for 15 min, cool in vertical position. For detecting indole either use Oxalate strips or add Kovacs’ reagent after suitable period of
39
incubation of stab-inoculated bottles. Uniform or brush like growth indicated motility and blackening of medium reveals production of H2S. Z.
MRS Lactobacillus agar (de Man, Rogosa & Sharp, 1960): Dissolve 10 g peptone, 10 g meat extract, 5 g yeast extract, 20 g glucose, 1 ml tween-80, 2 g K2HPO4, 5 g Na-acetate, 2 g Triammonium citrate, 20 mg MgSO4.4H2O and 50 mg of MnSO4.4H2O in 1 lt water and autoclave to sterilize at 121oC for 15 min. This medium can be gelled by adding 0.2% agar or can be used as basal medium for carbohydrate utilization by replacing glucose with any other sugar to be tested.
AA. Nitrate reduction test media: Nitrate broth: Nutrient broth with 0.1% potassium nitrate. Nitrate medium: Blood agar added with filter sterilized 5 ml of 20% potassium nitrate/ L.
AB. Nitrite Medium: Nutrient broth with 0.1% sodium nitrite.
AC. O/F test medium: Peptone 2g, NaCl 5g, K2HPO4 0.3g, 1%aqueous bromothymol blue 3ml, agar 3g, dissolved by boiling in 1 lt distilled water, pH is made 7.1, and autoclaved at 121oC for 15 min in 100 ml aliquots in flasks. Sugar is separately filter sterilized and added to 1% final concentration. Medium is filled in tubes to depth of at least 4 cm. Duplicate tubes of medium are inoculated by stabbing, one tube is quickly layered with sterile liquid petroleum to a depth of 1 cm and both tubes are incubated for up to 30 days.
AD. ONPG broth: Filter sterilize 250 ml of 0.6% ONPG (O-nitrophenyl-D-galactopyranoside) solution made in 0.01 M Na2HPO4 solution and add in to 750 ml autoclaved Peptone water, distribute in 2.5 ml volumes and store at 4oC for use within a month.
AE. Sodium potassium Magnesium (SPM) broth: For use as ONPG base instead of PW, SPM broth is made by dissolving 10 g Tryptone, 10 g NaCl, 1 g KCl and 4 g MgCl2. 6H2O in one lt water and sterilized by autoclaving.
AF. Phenolphthalein phosphate agar: Add 10 ml filter sterilized 1% aqueous phenolphthalein solution in molten1 lt NA cooled at 45oC, mix and pour the plates.
AG. Phenylalanine agar: Dissolve 1g L-phenylalanine, 3 g yeast extract, 1 g Na2HPO4, 5 g NaCl and 20 g agar in 1 lt water, boil to dissolve ingredients, tube the medium in 5 ml volume and sterilize at 115oC for 20 min. Allow to set the medium in long slants.
40
AH. Polyhydroxybutyrate agar: Dissolve 100 mg of sodium hydroxybutyrate (sodium salt of DLhydroxybutyric acid) in molten mineral base maintained at 60 oC and distribute in to Petri plates. Mineral base consist of 520 mg K2HPO4, 375 mg KH2PO4, 500 mg (NH4)2SO4, 50 mg CaCl2, 200 mg MgSO4.7H2O, 100 mg NaCl, 10 mg FeCl3.6H2O, 2 mg MnSO4.4H2O, 2 mg Na2MoO4.2H2O and 13 g of agar in 1 lt distilled water. Boiled to dissolve, dispensed in 20 ml aliquots and sterilized at 115oC for 15 min.
AI. Pyruvate fermentation medium: Dissolve 10 g tryptone, 5 g sodium pyruvate, 5 g K2HPO4 and 10 g agar in 1 lt water, adjust pH to 7.2-7.4 and add 10 ml 0.4% ethanolic solution of bromothymol blue. Dispense in to screw-capped tubes and sterilize at 115oC for 20 min.
AJ. Salt broth: Nutrient broth with 6.5% or desired concentration of sodium chloride.
AK. Selenite broth: To make base, dissolve 5 g peptone, 4 g mannitol, 4.3 g Na 2HPO4.12H2O, 2.8 g of NaH2PO4.2H2O, in one lt water with gentle heat and adjust pH to 7.2, dispense in 100 ml volumes, then sterilize at 121oC for 30 min. On day of use add 1 ml of filter sterilized 40% aqueous solution of sodium selenite aseptically in to 100 ml base and distribute in to 4-5 ml volumes for use.
AL. Soil Extract agar: Shifted (through No. 9 mesh sieve) and air dried garden soil (rich in nutrients and humus) is mixed well in 2.4 lt of water and autoclaved at 121 oC for 1 h. Filter to get clear extract. Add 5 g peptone, 3 g meat extract and 20 g agar, adjust pH to 7.0 and sterilize by autoclaving at 1150C for 20 min.
AM. Streptomyces extraction medium for preparing of Streptococcal group antigen: Dissolve 5 g NaCl, 2 g K2HPO4, 1 g MgSO4.7H2O, 40 mg CaCl2, 20 mg FeSO4. 7H2O, 10 mg ZnSO4.7H2O, 5 g yeast extract in 250 ml distilled water and boil 20 g agar in 750 ml of water, mix both and sterilize by autoclaving at 115 0C for 20 min. At the time of use melt the medium and add 25 ml of sterile 20% glucose and distribute in Roux bottles. AN. Todd-Hewitt broth: Soak 450 g of minced fat free meat in 1 lt of water and keep at 4oC overnight, skim the fat, heat at 85oC for 30 min, filter and adjust volume to 1 lt. Then to it, serially add to dissolve 20 g neo-peptone (Difco or Evans’ peptone, these peptones not encourage production of proteinases which may destroy M protein), 2.7 ml of 10 N-NaOH, 2 g
41
NaHCO3, 2 g NaCl, 1 g Na2HPO4.12H2O, 2 g glucose. Adjust pH to 7.8 and sterilize by autoclaving at 1150C for 20 min.
AO. Media for antigen extraction from Streptococci for grouping: Lancefield method; Re-suspend pellet from 50 ml Todd Hewitt broth in 0.4 ml of 0.5 M HCl. Boil for 10 min in water bath, cool and add one drop of 0.02% phenol red. Neutralize with 0.2 M NaOH and centrifuge to collect supernatant extract. Formamide method of Fuller; re-suspend pellet from 5 ml glucose broth in 0.1 ml formamide, heat in oil bath at 160oC for 15 min. cool and add 0.25 ml acid alcohol (0.5% HCl and 95% ethanol). Shake and centrifuge, add 0.5 ml acetone to supernatant, shake and centrifuge, discard supernatant and dissolve precipitate in 0.3-0.4 ml NSS, add a drop of phenol red and neutralize with 0.5 M NaOH. Alkaline pH increases possibility of cross-reaction.
AP. TSI (triple sugar iron) agar: Boil to dissolve 3 g meat extract, 3 g yeast extract, 20 g peptone, 1 g glucose, 10 g lactose, 10 g sucrose, 200 mg FeSO4.7H2O or 300 mg of Ferric citrate, 5 g NaCl, 300 mg Na2S2O3.5H2O and 20 g agar in 1 lt water. Adjust pH to 6.8, add 12 ml of 0.2% aqueous phenol red, distribute in screw-capped tubes in 7 ml volume and sterilize by autoclaving at 1150C for 20 min. Allow to cool in slanted position to have good long 3 cm butt and about 1” slant.
AQ. Tyrosine hydrolysis agar: Take 100 ml of salt free NA base, melt and suspend 500 mg of tyrosine and sterilize by autoclaving at 1150C for 20 min, then pour the plates.
AR. Urea medium: a. Christensen’s medium: Dissolve (through gentle heating) 1 g peptone, 5 g NaCl, 2 g KH2PO4 and 20 g agar in 1 lt water, adjust pH to 6.8 and sterilize by autoclaving at 115 0C for 20 min. Allow to cool to 60oC and add 6 ml of filter sterilized 0.2% phenol red containing 1 g of glucose and 50 ml of 40% filter sterilized urea solution. Distribute aseptically in vials and cool to set in slopes. b. Christensen’s broth medium: Dissolve 1 g glucose, 1 g peptone, 5 g NaCl, 1.2 g Na2HPO4, 0.8 g KH2PO4 in one lt water, adjust pH to 6.8 and add 5 ml of 0.2% aqueous phenol red and sterilize by autoclaving at 115 0C for 20 min. Cool to 55oC and add filter sterilized 50 ml of 40% urea solution and distribute in suitable sterilized vials aseptically.
42
AS. Xanthine and Hypoxanthine agar: Same as tyrosine hydrolysis medium except the tyrosine replaced by xanthine or hypoxanthine @ 0.4-0.5% level.
27. SOME BASIC MEDIA USED IN MICROBIOLOGY: a. Nutrient Broth (NB): Meat Extract 10g, Peptone 10 g NaCl 5g, water to 1 lt. Adjust pH to 8.0-8.4 boil to dissolve and cool to readjust pH to 7.2-7.4, autoclave to sterilize at 115oC for 20 min. b. Nutrient agar (NA): NB with 2% agar.
c. Semisolid NA: NB with 0.4% agar.
d. Peptone water (PW): Peptone 10 g, NaCl 5 g, water to 1 lt. Adjust pH to 8.0-8.4, boil to dissolve and cool to readjust pH to 7.2-7.4, autoclave to sterilize at 115oC for 20 min.
e. PW agar: PW with 2% agar. f. Robertson’s Cooked Meat Medium (RCM): One Kg of minced meat is boiled for 20 min in 1 lt 0.05N NaOH. Skim the fat and adjust pH to 7.5. Strain through gauze and dry the meat at 50oC. Take dried meat pieces up to 1-inch height and add NB to give depth of about 5 cm. autoclave to sterilize at 115oC for 20 min.
g. Brain heart infusion broth (BHI): Take 225 g each of minced heart and minced brain tissue (devoid of fat) and mix in 1 lt water, keep for infusion overnight at 4 oC, skim the fat, add 10 g of peptone and 5 g of NaCl, boil for 30 min. Filter and adjust pH to 8.4, boil for 20 min and cool to readjust pH to 7.6, autoclave to sterilize at 115oC for 20 min.
h. BHI Agar: BHI broth with 2% agar.
i. Mueller Hinton agar: Meat infusion 6g, casein hydrolysate 17.5 g, starch (soluble) 1.5 g, agar 10 g, and water to 1 lt. Adjust pH to 7.4, autoclave to sterilize at 121oC for 15 min. j. Blood agar (BA): Add 50 ml defibrinated blood to sterilized 950 ml NA cooled to 50 oC then plated.
43
k. Layered BA: It is better for observation of haemolysis; first the plates are made of PW agar and when set layer of BA is pored over PW agar. l. Chocolate agar: Place a BA plate at 65oC for 1-1.5 h or instead of mixing blood at 50oC keep NA on 80oC for some time after mixing 5% blood (V/V).
m. Serum agar: Nutrient agar with 5% (V/V) serum. Serum is added in to sterilized NA at 5055oC.
n. Serum glucose agar: Same as serum agar but 20% glucose (filter sterilized) and serum are added @ 5% (V/V) each.
o. Fildes’ agar and broth: In NA/ NB Peptic digest of blood is added to the extent of 2-5% after heating, to remove chloroform. It stimulates the growth of Haemophilus.
p. Glucose broth: NB with 20% sterilized glucose solution added to the extent of 5 % (V/V). q. Glycerol broth: NA with 5-7% (V/V) glycerol, autoclave to sterilize at 115oC for 20 min.
r. Mac-Conkey broth double strength: Peptone 40 g, sodium taurocholate 10 g, lactose 20 g, NaCl 10 g, bromocresol purple (1% w/v ethanolic solution) 2 ml in one lt distilled water, pH to 7.4. Sterilized at 115oC for 10 min.
s. Laural tryptose broth, double strength: Tryptose 40 g, lactose 10 g, NaCl 10 g, K2HPO4 5.5 g, KH2PO4 5.5 g, Sodium lauryl sulphate 0.2 g in 1 lt distilled water, pH 6.8. Sterilized at 115oC for 10 min.
t. Brilliant green lactose bile broth: Peptone 10 g, lactose 10 g, ox bile 20 g, brilliant green (0.1% aqueous solution) 13 ml in 1 lt distillied water, pH 7.0-7.5. Sterilized at 115oC for 10 min. u. Tryptone water: Tryptone 20 g, NaCl 5 g in 1 lt distilled water, pH 7.5, Sterilized at 115 oC for 10 min.
44
v. Membrane lauryl sulphate broth: Perptone 40 g, yeast extract 6 g, lactose 30 g, Sodium lauryl sulphate 1 g in 950 ml distilled water, pH 7.4, add phenol red (0.4% aqueous solution) 50 ml . Sterilized at 115oC for 10 min.
28. MEDIA FOR DETECTING PIGMENT PRODUCTION ABILITY OF BACTERIA a. Chromobacterium: Mannitol Yeast extract agar, incubated at 37oC for 24 h and then at RT for 5 days. (Elimination of meat extract and addition of mannitol improve colour production by Chromobacterium and Xanthobacterium).
b. Mycobacterium: LJ slope medium, continuous light exposure increases colour formation.
c. Pseudomonas: For detection of pyocyanin (blue non fluorescent, water and chloroform soluble) and pyoverdin (yellow, fluorescent, water soluble but not in chloroform, require phosphate for formation) use King’s medium A and B, respectively. Incubation at 37 oC for 24-96 h or incubate at 37oC for 24 h and then at RT for 3 days. Or Use Kligler iron agar, it enhance colour production by P. cepacia. Wide grades of pigment colours can be observed varying the carbon source in the medium. Pyocyanin can be seen on PIA (Pseudomonas isolation agar) but King’s medium B is the best. Pseudomonas aeruginosa may produce red (pyorubin) or dark-brown (pyomelanin) besides pyocyanin. Pyocyanin may turn red or brown on prolonged exposure to air. Growth in1% DL-glutamate allows formation of pyorubin but not pyomelanin while minimal-salt medium supplemented with 1% tyrosine enhances pyomelanin production. Furunculosis agar (tryptone 10g, yeast extract 5g, Ltyrosine 1g, NaCl 2.5g, agar 15g/ lt) can also b used for detection of pyomelanin production. d. Serratia marcescens: Nutrient agar or Mannitol yeast extract agar or King’s medium, incubated at 22-30oC.
e. Micrococcus spp.: Potato slopes. f. Melaninogenicus oralis: Blood agar. Due to formation of protohaemin, colonies’ red fluorescence under UV is due to protoporphyrin, a precursor of protohaemin.
g. Clostridium difficile: Yellow fluorescence is emitted from colonies grown on blood agar under long-wave UV.
45
h. Potato slopes: Clean and scrub potato, cut cylinders with a 18-20 mm cork borer. Cut each cylinder obliquely in to 2 and place one half in to 30 ml potato tube. Fill with distilled water and steam for 30 min; pour off water and then autoclave at 115 oC for 20 min.
i. Mannitol Yeast extract agar: Peptone 2.5 g, NaCl 2.5 g, mannitol 5.0 g, yeast extract 2.5 g, agar 20 g, distilled water to make 1 lt. boil to dissolve, adjust pH to 7.0, and autoclave at 115oC for 20 min. j. King’s medium for pyocyanin: Peptone 20 g, glycerol 10 g, K2SO4 10 g, MgCl2 1.4 g, agar-agar 20 g, water to 1 lt, pH 7.2 autoclave at 115oC for 10 min. k. King’s medium for fluorescine: Protease peptone 20 g, glycerol 10 g, K2HPO4 1.5 g, MgSO4 7H2O 1.5 g, agar-agar 20 g, water to 1 lt, pH 7.2 autoclave at 115oC for 10 min.
29. Mc-FARLAND STANDARD PREPARATION McFarland
1%
aqueous 1%
standard
Sulfuric acid in Barium
aqueous
Density
of
International units
chloride
bacteria
in of opacity
ml
in ml
millions/ ml
0.5
9.95
0.05
150
1
9.9
9.1
300
3
2
9.8
0.2
600
7
3
9.7
0.3
900
10
4
9.6
0.4
1200
12
5
9.5
0.5
1500
15
6
9.4
0.6
1800
18
7
9.3
0.7
2100
20
8
9.2
0.8
2400
24
9
9.1
0.9
2700
27
10
9.0
1.0
3000
30
Make solution in chemically resistant glass of high quality and transparency. Plug the tube and seal with wax or seal the tube with flame. Density of bacteria varies from strain to strain; therefore it is always advised to standardize the tubes for your strain.
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30. SOME IMPORTANT PARAMETERS FOR EXPERIMENTAL ANIMALS USED IN MICROBIOLOGY: Parameters
Rabbit
Guinea-
Mouse
Rat
Hamster Fowl
pig Rectal temperature in oC
37-39
37-39
35-39
36-40
36-39
40
Respiratory rate/min
35-60
90-150
100-250
70-150
30-140
15-35
Heart rate/min
205-235
130-190
500-600
260-450
350
300
RBC count, 106/mm3
4-7
4-7
7-11
7-10
4-10
2-4
Haematocrit, %
30-50
35-42
35-45
35-45
39-60
25-45
Blood volume, ml/kg body
70
75
80
50
78
60
Gestation in days
31
68
19-21
20-22
15-18
21, incubation
Litter size (mean)
6
3
8
8
7
Litters per year
4
3
9
9
4
Weaning age in days
42-56
2-18
19-21
20-22
20-22
Birth
50
85
1-1.5
4-5
1.5-2.5
4 weeks
600
240
15
45
35
8 weeks
1300
450
20-25
200
75
12 weeks
2100
600
25-30
300
90
20 weeks
2700
900
25-35
450
120
36 weeks
5000
1000+
25-35
900
weight
Body weight in grams at age of
31. SIZES OF HYPODERMIC NEEDLES USED IN MICROBIOLOGY Metric, in mm
Imperial
Hub colour
0.5×10
25G×3/8inch
Orange
0.5×25
25G×21/16 inch
Orange
0.63×30
23G×19/16inch
Blue
0.8×40
21G×3/2 inch
Green
1.1×50
19×2 inch
White
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32.
PRECIPITATION
OF
PROTEIN
ANTIGENS
WITHOUT
DENATURATION A. Salting out with Ammonium Sulphate Ammonium Sulphate gives a slightly acidic pH thus those antigens can tolerate slight acidity should be precipitated with it. For precipitation a 50 mM buffer in range of 6-7 pH should be used. How much Ammonium sulphate is required: While adding ammonium sulphate there is increase in volume of the solution therefore amount of ammonium sulphate should be calculated considering that increment in the volume with following formula: g of ammonium sulphate required for 100 ml= 53.3(C2-C1)/ 100-0.3 C2 Where C1 is the initial % concentration in solution and C2 is the required % concentration of ammonium sulphate. This equation calculate requirement at 20 oC and allows for the increase in volume. Salting out can be carried out with sodium citrate if precipitation has to carry out at higher pH.
B. Precipitation with water miscible organic solvent as ethanol and acetone Volume of solvent required for 100 ml= 100(C2-C1)/ 100-C2 Where C1 is the initial % concentration and C2 is the required % concentration of the solvent. Organic solvents have lower specific gravity thus precipitate settle rapidly.
C. Precipitation with water miscible organic polymers as polyethylene glycol (PEG) 6000 or 20000 PEG removal from precipitate requires ultra-filtration or dialysis and usually 20 kDa, usually 10% TCA (v/v) is
48
sufficient, for lighter proteins up to 20% TCA may be required. TCA can be removed from pellet by repeated washing with distilled water. c. Organic Solvents: Four volumes of acetone or 9 volumes of ethanol are required. Usually solvent is mixed at RT and then cool the mixture to -20oC for at least an hour prior to centrifugation.
34. REAGENTS REQUIRED FOR DIFFERENT BACTERIOLOGICAL TESTS: a. Phenylalanine test reagent: Acidic Ferric chloride (12.0 g of FeCl3.6H2O dissolved in 100 ml water containing 2.5 ml concentrated HCl).
b. Gelatinase and caseinase test reagents: A. Acidic Mercuric chloride: Dissolve 12 g of HgCl2 in 80 ml distilled water plus 16 ml HCl). B. 30% Trichloroacetic acid in water. c. MR Reagent: Dissolve 40 mg methyl red in 40 ml ethanol and then make the volume to 100 ml.
d. Nitrate test reagent: Solution A: 0.33% sulphanilic acid in 5N acetic acid. Solution B: 0.5% α-naphthylamine in 5% acetic acid or 0.13% α-naphthylamine-7sulphonic acid in 5N acetic acid. Reagent C: Zinc dust. e. VP Test reagents: 1% creatinine solution in 0.1N HCl; 5% α-naphthol in 95% ethanol; 40% aqueous KOH. i.
O’Meara Method: After MR test add 0.05 ml of 1% creatinine solution and 1 ml of 40% KOH, shake well and keep in slanting position, eosine pink colour appear in 1-4 h.
ii.
Berritt method: After doing MR test add 0.6 ml of 5% α-naphthol made in 95% ethanol and 0.2 ml of 40% KOH, mix well ands keep standing for 15 min, appearance of red colour indicate positive reaction.
f. Indole Test: i.
Kovak’s reagent for Indole test: 5.0 g p-di-methyl-amino-benzyladehyde dissolved in 75 ml amyl alcohol and 25 ml of concentrated HCl is added slowly and stored in amber coloured bottles in dark.
49
ii.
Ehrlich’s reagent for indole test: Dissolve 1 g p-di-methyl-aminobenzaldehyde in 95 ml of absolute ethanol and then 20 ml of concentrated HCl is added slowly and stored in amber coloured bottles in dark.
g. Benedict’s reagent for presence of reducing sugars: 17.3 g Sodium citrate, 10 g Sodium carbonate, 1.73 g CuSO4. 5H2O. Sodium citrate and carbonates are dissolved in 60 ml distilled water and copper sulphate in 20 ml of distilled water, mixed together and volume is made to 100 ml.
h. Acid mercuric chloride for proteinases: Induce precipitation of proteins and used to detect proteinases (gelatinase, caseinase) activity of bacteria. Dissolve 12 g of mercuric chloride in 80 ml water and then add 16 ml of concentrated HCl drop-by-drop. 30% trichloroacetic acid may also be used instead of mercuric chloride solution.
i.
Nessler’s reagent: For detection of ammonia. Dissolve 5 g KI in 5 ml distilled water and then add cold saturated HgCl2 solution till a slight precipitate appears and persist even after shaking. Add 40 ml 9N-NaOH and dilute to 100 ml with water. Use after 24 h of standing. Alyternatively it can be made by dissolving 8 g of KI and 11.5 g of HgI 2 in 20 ml water make it 50 ml with water and then add 50 ml of 6 N-NaOH, store for 24 h before use.
35.
BACTERIOLOGICAL
TEST
STRIPS
IMPREGNATED
WITH
REAGENTS a. For Detection of H2S: Impregnate the Whatman paper no. 1 strips in hot saturated Lead acetate aqueous solution (10%).
b. For Indole test:
Impregnate the Whatman paper no. 1 strips in hot saturated Oxalic acid.
Impregnate the Whatman paper no. 1 strips in 10% dimethylaminobenzaldehyde, 20% phosphoric acid made in methanol.
c. PPA test (Production of phenylpyruvic acid from phenylalanine): Impregnate the Whatman paper no. 1 strips in 0.5% phenylalanine in PBS pH7.2.
d. For H2O2: Impregnate the Whatman paper no. 1 strips dipped in peroxide reagent (BDH).
50
e. For Oxidase test: Impregnate the Whatman paper no. 1 strips in solution made from a grain of N-N-N-N-tetramethyl-p-phenylenediamine dihydrochloride made in water containing 1% ascorbic acid.
f. O/129 discs for Vibrionaceae: Impregnate the Whatman paper no. 1 discs in 0.1% solution of 2-4-diamino-6,7-di-isopropyl-pteridine phosphate in acetone. It inhibits the growth of most vibrios. Discs containing 150 μg of O/129 are used for primary differentiation; discs containing 10 μg of O/129 have differential value for various Vibrio spp.
g. Optochin discs: Impregnate the Whatman paper no. 1 discs in 0.05% ethyl hydrocuprein hydrochloride aqueous solution. Pneumococci is more sensitive to optochin than other streptococci to yield a clear zone of about 14 mm while others yield zone 20 h or culture contaminated with fibrinolytic bacteria e.g. streptococci.
Coagulating plasma with calcium can make serum from citrated plasma salt.
f. Rapid coccal transformation: Exposure of Campylobacter and Helicobacter strains to aerobic environment lead to formation of coccal forms from bacillary forms grown microaerobically. For the test two plates are of suitable medium inoculated with test organism and incubated for 24 h. One plate is taken out of micro-areophilic environment and incubated aerobically at RT for 24 h. Smears are made from both of the plates and examined after staining. Helicobacter strains turns to coccal forms within an hour of aerobic exposure.
g. Decarboxylase test: Take growth from NA slants, wash with water and suspend to about 109 cfu/ml in water. Add 40 μl each of culture suspension, 0.3M solution of test amino acid (pH 5.0) and 0.01% bromocresol purple made in 0.0125 M PBS, pH 5.0, incubate at 37oC in water bath and observe after 2, 4 and 24 h. Colour change from yellow to blue indicate decarboxylation of amino acid.
h. DNase test: DNA toludine blue agar plates are spot inoculated with test organisms and incubated micro-aerobically, zones of clear colourless or of pinkish hue around the bacterial growth is indicative of DNase production. DNase medium: Tryptose 20 g, DNA 2 g, NaCl 5 g, Toludine blue 0.1g, agar-agar 20 g, in 1000 ml distilled water. Sterilized through autoclaving. Or: DNA 0.3 g, 0.01 M CaCl2 1 ml, NaCl 10 g, agar-agar 6.5 g, dissolved in 0.05 M Tris HCl (pH 9.0) to make it 1 lt. Boil until DNA is dissolved, cool to 50 oC and add 2.5 ml of 3% toludine blue O (filter sterilized), aliquot in 1 ml tubes, inoculate with test colony and observed for 1-3 days for development of pinkish colour in tube.
i. Gelatinase test: Gelatin agar plates are spot inoculated with test organism and incubated for 3 days at 37oC. Flood the plates with 30% TCA, clear zone around colonies of bacteria indicate positive test. Gelatin agr is made by dissolving 4 g of gelatin in 50 ml water by boiling and then making it 1 lt with nutrient agar, then autoclaved to sterilize and plates are poured.
53
j. Hippurate Hydrolysis test for Enterobacteriaceae: Suspend one loopful of 18-24 h growth of test bacterium from blood agar plate in 0.4% of 1% sodium hippurate in PBS pH 7.0 (made fresh or stored frozen at –20oC). Incubate for 2 h at 37oC then slowly add 0.2 ml of freshly made 3.5% Ninhydrin (in 1:1 acetone and butanol) made freshly or stored frozen at –20oC. Reincubate for 10 min as above. Development of deep purple colour indicate positive test. Klebsiella gives positive and Enterobacter yields negative results. k. Indole test: Take growth from NA slants, wash with water and suspend to about 10 9 cfu/ml in water. Add 40 μl of culture suspension in to 60 μl of 0.1% tryptophan made in 0.025 M PBS pH 8.0. Incubate for an hour at 37oC in water bath. Add 60 μl of Kovac’s reagent made in iso-amyl alcohol. Red colour appears immediately in positive cultures. l. Gluconate oxidation test: Inoculate gluconate broth and incubated at 37 oC for 2 days. Add 1 ml of Benedict’s solution to 5 ml culture, boil for 10 min. Formation of brown/orange/tan precipitate constitute positive test.
m. Gluconate utilization broth: Peptone 1.5 g, yeast extract 1 g, K2HPO4 1 g, potassium gluconate 40 g or sodium gluconate 37.25 g make volume to 1 lt with water, pH 7.0. Sterilize at 115oC for 20 min.
n. Malonate Utilization: Take growth from NA slants, wash with water and suspend to about 109 cfu/ml in water. Add 40 μl of culture suspension in to 40 μl each of 1% aqueous sodium malonate and 0.025 M PBS pH 6.0 containing phenol red indicator. Incubate for 24 h at 37oC in water bath. Appearance of red colour indicate positive test.
o. Niacin (nicotinic acid) test: Niacin is produced by Mycobacteria. Grow the test organism for 30 days in LJ medium and harvest the growth in 0.3 ml sterile water. Autoclaved for 30 min, 40 μl of it is put in a tile, add 40 μl each of 1.5%ethanolic-O-toludine and 10% of aqueous cyanogens bromide. Appearance of pink or orange colour indicate a positive reaction.
p. ONPG (o-nitrophenyl-β-D-galactopyranoside) test: Used to detect potential lactose fermenters, lactose fermentation is dependent o two enzymes, β-galactosidase which attacks lactose and a permease, which regulatespenitration of lactose in to cells. The simple
54
test for β-galactosidase is to take growth from NA slants, wash with water and suspend to about 109 cfu/ml in water. Add 40 μl of culture suspension in to 40 μl of ONPG made in 0.01 M Na2HPO4 pH 7.5 (can be store in dark and cool place). Incubate at 37 oC and examined for development of yellow colour (colour less ONPG is converted in to yellow onitrophenol after 1, 2 and 24 h for positive reaction. It is good test for late lactose fermenters, for those an alternate test is to use 5-10% lactose in medium for carbohydrate fermentation.
q. Oxidase test (Cytochrome oxidase): Impregnate the Whatman paper no. 1 strips in aqueous solution of 0.1% N-N-N-N-tetramethyl-p-phenylenediamine dihydrochloride (instead of tetramethyl, 1% dimethy i.e., p-aminodimethylamine oxalate is more stable and gives equivocal results) 10 ml water and allow the strip to half dry and put on a galls slide on plate. Take a loop-full of bacterial growth from sugar and blood free agar medium either with platinum loop or glass rod or with sterilized tooth prick (wooden stick) and rub it on the strip. Deep purple pink colour appears within 30-60 seconds in positive case.
r. Catalase test: Put a drop of 3% H2O2 on clean glass slide and then pick up test colony with platinum loop or with wooden stick from blood and sugar free medium, mixing of bacterial growth with H2O2 results in bubbling or frothing indicates the positive reaction.
s. Phenylalanine test: Phenylalanine in converted by some bacteria in to phenyl pyruvic acid (PPA) which can be detected by adding ferric chloride in the same way as most other keto acids. Take growth from NA slants, wash with water and suspend to about 10 9 cfu/ml in water. Add 200 μl each of bacterial suspension and 0.2% DL-phenylalanine solution in saline; incubate for 4 h at 37oC. Then add a few drops of 10% H2SO4 with phenol red indicator and then 4-5 drops of half saturated aqueous FeSO4(NH4)2SO4.6H2O. Appearance of green colour indicates positive reaction. Green colour fades rapidly so read the reaction quickly. or add 40 μl each of bacterial suspension, 0.03 M phenylalanine, 0.025 M PBS pH 6.8, incubate for 2 h at 37oC in water bath. Add 40 μl of 2% aqueous ferric chloride, green colour indicate production of phenylpyruvic acid.
55
t. Levon/ Dextran production test: Inoculate test culture on sucrose medium (NA with 1020% sucrose). Levon producer (Streptococcus salivaris) produces large mucoid colonies while dextran producers (Streptococcus sanguis) produce small glossy colonies.
u. Porphyrin test (for determining requirement of factor X): Take a loopful growth of test organism from chocolate agar and inoculate in to 0.5 ml substrate solution (2 mM δaminolaevulinic acid, ALA; 0.08 mM MgSO4 in 0.1M PBS, pH 6.9). Incubate for 4 h at 37oC. Observation of fluorescent red colour under Wood’s lamp (360 nm) or examine after 24 h on adding 0.5 ml of Kovac’s reagent for development of red colour. v. Phosphatase test: Used for identification of pathogenic Staphylococcus. Spot inoculate phosphatase test agar (NA with 1% phenolphthalein phosphate added at 50oC) and incubate at 37oC for 18 h. Put 0.1 ml ammonia solution in a lid of plate and keep the plate inverted on lid. . Free phenolphthalein released due to presence of phosphatase reacts with ammonia to form bright pink colour in positive colonies.
w. Tween hydrolysis: Spot inoculate Tween 20 or 80 (oleic acid ester of poly-oxyalkylene derivative of sorbitan) medium with the test strain and incubate at optimum growth temperature for 24 h or more and observe for opaque (opacity is due to crystal formation of lipolysed sorbitan) halo indicating hydrolysis of Tween. Tween Medium: Peptone 10 g, NaCl 5 g, CaCl2.2H2O 0.1 g, agar-agar 20 g, make it 1 lt with distlled water, adjust pH to 7.4 and autoclave to sterilize, cool to 40-50oC and add 10 ml of sterilized (autoclaved separately in 5 ml aliquots at 121 oC for 15 min) Tween, dispense in petri-plates. Alternatively test can be done in broth medium (a solution of 0.5% Tween 80 or Tween 20 in PBS pH 7.0 having neutral red indicator (20 ml of 0.1%/ L) is distributed in 2 ml volume and autoclaved to store at 4oC in dark in screw-capped vials, colour of medium is amber). (Pseudomonas aeruginosa and Mycobacterium kansasii are used as positive control). x. Urease test: For Routine test Christensen’s urea medium is used. On hydrolysis of urea some bacteria produce ammonia, which alkalinize the media. For rapid test, take growth from NA slants, wash with water and suspend to about 10 9 cfu/ml in water. Add 40 μl of bacterial suspension in to 60 μl of 2% urea solution and 60 μl of 0.025 M PBS, pH 6.0 with phenol red. Incubate for 60 min at 37oC in water bath and examine every 15 min for appearance of red colour, negative samples should be incubated for 4 h.
56
y. X & V Factor requirement test: Inoculate blood agar and nutrient agar plates with test organism and spot inoculate Staphylococcus aureus in streaked area on each of the plate. Incubate appropriately and observe for growth and satellite colonies. Growth on blood agar only indicate fastidiousness for X factor, satellite colonies only on blood gar plate indicate requirement of X & V factors, Satellite colonies on both plates indicate requirement for only V factor. Growth on both plates without satellite colonies indicate neither X nor V factor requirement. Alternate test: Inoculate test organism in separate tubes containing glucose peptone with bromothymol blue and supplemented with X or V or both X & V factors, incubated and observed for colour change from blue to yellow.
z. Carbon Dioxide requirement for Bacterial Growth (Candle Jar): Some capanophilic (CO2 loving) bacteria require increased tension of CO2 but not inhibited by the presence of O2 as many of the anaerobes do. To provide excess CO2 in environment many methods are used as kits available from Oxoid and BD, they may give CO2 concentration up to 10%, another way is to evacuate the Filde’s Jar or McIntosh Jar to about 2/3 and then replenish vacuum with CO2 or Mixture of CO2, N2 and H2. The other simple method is Candle jar method, however many bacteriologist objects to it because of being ill defined environment, presence of many fumes generated due to burning of candle some of which may be affecting the bacterial growth. In Candle jar, CO2 concentration is about 2.5% and O2 is 17%. Gas generating kits usually produces atmosphere of 10% CO2 and H2 and absence of oxygen. aa. Nitrate reduction test: Bacteria may reduce Nitrate in to various products, e.g., nitrite, hyponitrite, hydroxylamine, ammonia, nitrous oxide and nitrogen. Therefore, to say nitrate reduction first step is to detect nitrite, if it is absent then test for nitrate to confirm that nitrate is not further broken. To detect presence of nitrate Zinc dust is used to reduce the nitrate in medium to nitrite, which is then detected as usual, if bacteria reduce nitrate than test should be negative after adding zinc dust. The other way is indirect, all nitrate-reducing bacteria reduce haemoglobin in Blood agar to methaemoglobin and test is positive even when nitrate is reduced below nitrite, nitrite disc (KNO 3). Durham’s tube can be put in to nitrite or nitrite broth, if it is reduced to N2 then gas can be detected in tube. In nitrite broth and nitrate broth use 0.01% NaNO3 or KNO3 in peptone water. Reduction can be detected using a few drops each of nitrate reagent A and B after suitable incubation time, which detects presence of nitrite. Interpretation of test: Culture
Test used
Results
Interpretation
57
medium Nitrate broth
1.
For
nitrite,
using No
reagents A and B
red Nitrite absent, check for nitrate
colour Red colour
Nitrite broth
Nitrate is reduced to nitrite
2. For Nitrate with Zn dust No red color
Nitrate is broken below nitrite
and reagents A and B
Nitrate is not reduced
3.
For
nitrite
reagents A and B
Red colour using Red colour No red color
Nitrite not reduced Nitrite is reduced
ab. Temperature tolerance and growth temperature: Most vegetative bacteria are killed at 56oC in 30 min but a few as Staph. aureus, Aerococcus viridans and Enterococci, survives for 30 min at 60oC and this method is used to select these. Most pathogens grow at 35-40oC but tolerate and can grow out side this temp range. However, Neisseria gonorrhoeae dies rapidly outside this temperature range. Listeria and Yesinia spp. can grow well below 4 -5oC, which can used to enrich these pathogens. In Most laboratories, ability of bacteria to grow at 20-22, 30, 37 and 44- 45oC is used to differentiate between genera and species of various pathogens.
ac. Co-aggregation test: Some bacteria as Actinomyces viscosus produces surface fibrils of other structures which act as adhesins for other bacteria and cause agglutination called coaggregation. Some bacteria cause agglutination of host cells as of RBCs and then it is called haem-agglutination.
ad. Carboxymethyl cellulose hydrolysis test: Test is used to deifferentiate between Bacillus subtilis from related species (B. subtilis and B. licheniformis are positive and B. amyloliquefaciens is negative). NA plates are made with 0.5% (w/V) carboxy-methylcellulose and spot inoculated. Incubated for 48 h at 30-37oC. Then Flood the plates with 2M HCl for 10 min. Pour off the HCl and then flood with ethanol, keep at RT for 8-10 h or until white precipitate is formed.
ae. PYRase test: Enterococci and group A Steptococci gives this test positive by hydrolyzing L-pyrrolidonyl-B-naphthylamide (PYR). To test, dissolve 25 mg of PYR in 1 ml methanol and then add 100 ml distilled water. Pick up two colonies on a swab and moisten with PYR and hold for 10 min and add one drop of cinnamaldehyde reagent. Development of red colour in 15 min in positive reaction and no colour in 30 min is negative.
58
af. Benzidine Test: Reagents: The benzidine solutions were prepared by the method of Bing and Baker (1931; also see Ham, 1953). One gram of benzidine-2 HCl (Fisher) or benzidine base (Merck) was partially dissolved in 20 ml of glacial acetic acid, 30 ml of distilled water were added and the solution heated gently, cooled, and 50 ml of ethyl alcohol (95 per cent) were added. The development of a slight yellow color in the dihydrochloride upon storage does not alter the sensitivity of the reagent (Ham, 1953). At refrigerator temperatures the reagent is stable for at least 1 month. Fresh hydrogen peroxide solutions were prepared each week by diluting 30 per cent reagentgrade hydrogen peroxide (Merck). Methods of performing the benzidine test. After good growth of the respective organisms was obtained (24 to 48 hr) the plate was flooded with the benzidine dihydrochloride solution followed by the addition of an approximately equal volume of 5.0 per cent hydrogen peroxide. The benzidine dihydrochloride reagent must come in contact with all of the microbial growth before the introduction of the hydrogen peroxide. If the culture in question possessed ironporphyrin compounds, a blue-green to deep blue coloration of the microbial growth promptly developed. Some variation in the intensity of the color was noted depending on the species and strain examined. Only the microbial growth (either individual colonies or confluent growth) evidenced a positive test. The medium, itself, never gave a positive reaction unless high concentrations of iron were added. The benzidine test, as described by Bing and Baker (1931), for the occult blood determination employed 0.6 per cent concentration of hydrogen peroxide. However, preliminary studies showed that 4 to 6 per cent peroxide gave much more rapid and intense color development. All attempts to perform the benzidine test directly on broth cultures failed. This may have been due to an actual dilution of the ironporphyrin compounds in the culture, a dilution of the reagents, or a masking effect due to the color of the medium. However, if cultures of benzidine-positive organisms were first centrifuged and the bacterial sediment resuspended in 0.5 ml of the reagent followed by the addition of 0.5 ml of 5.0 per cent hydrogen peroxide, a positive test could be obtained.
37. STAINS AND REAGENTS USED IN MICROBIOLOGY a. Acetone-Iodine solution: Mix 3.5 ml of strong iodine solution in 96.5 ml acetone.
b. Strong Iodine solution: Dissolve 10 g Iodine and 6 g Potassium iodide in 10 ml water and adjust volume to 100 ml with 90% ethanol.
59
c. Acid Alcohol: Add drop-by-drop 3 ml concentrated HCl in 97 ml of 95% ethanol and then mix well. d. Albert’s Stain: Dissolve 200 mg of Malachite green and 150 mg of Toluidine blue in 2 ml of 95% ethanol. Mix 1 ml acetic acid in 97 ml distilled water. Mix both acidic water and dye solution, allow to stand at RT overnight and then filter to clear the stain.
e. Ammonical Silver Nitrate: Dissolve 5 g of Silver nitrate in 100 ml distilled water. Take 90 ml of solution and add drop-by-drop strong solution of ammonia (sp. gravity 0.88) until the ppt forms but just dissolves on shaking. Thereafter add remaining silver nitrate solution drop-by-drop until solution become a little turbid and remains turbid even after shaking. Store in cool and dark place.
f. Ammonium oxalate crystal violet stain: Dissolve 2 g crystal violet in 20 ml of 95% ethanol and make it 100 ml with 1% aqueous ammonium oxalate. g. Solutions of different dyes/ stains: Stain
Concentration
Stain
Concentration
Bismarck
0.2% aqueous
Malachite
0.5
green
aqueous
brown
or
Chrysoidin
0.4% aqueous
Safranine
0.5% aqueous
Crystal
0.5-2%, in 90% ethanol,
Leishman’s
0.15%
violet/methyl
dissolve dye in ethanol then
stain
Leishman’s
violet
dilute to make volume.
powder
5%
in
methanol
h. Aqueous iodine (BP): In 20 ml of water dissolve 10 gm potassium iodide and then 5 g iodine and make the volume to 100 ml with water.
i. Liquor iodi fortis (BP): In 10 ml of water dissolve 10 gm potassium iodide and then 6 g iodine and make the volume to 100 ml with methylated spirit.
j. Carbol Fuchsin: Dissolve 15 g of Carbol fuchsin in 85 ml of phenol and make it to 1600 ml with 16.67% ethanol or 16.67% propanol. Filter and keep in airtight-stoppered bottle. It can be diluted suitably (10-20 times) to make weak carbol fuchsin solution.
60
k. Giemsa Stain stock: Dissolve 1 g Giemsa powder in 60 ml hot (60oC) glycerol and maintained hot for 2 h. Cool to RT and add 60 ml methanol and keep in airtight-stoppered bottle for 2 weeks for maturation. Matured stain is diluted 1:10 in 0.01 M PBS (pH 7.0) for making working solution, which can be used after 30 min. l. Kirkpatrick’s fixative: Mix 60 ml of absolute ethanol with 30 ml of chloroform and 10 ml of 40% formaldehyde (formalin). m. Loeffler’s Methylene blue (Polychrome methylene blue): Mix 30 ml of 1% ethanolic solution of methylene blue in 100 ml of alkaline distilled water (0.01% KOH in water). Store at RT in cotton plugged half full bottle at least for few months with intermittent shaking for maturation of stain. For good quality maturation storage of 10 years is recommended, such matured stain is called polychrome methylene blue. n. Lugol’s Iodine: Add 20 ml of aqueous iodine in 80 ml of water. o. Albert’s Iodine: Dissolve 9 g potassium iodide in water and then dissolve 6 g of iodine and make th volume to 900 ml with water.
p. Iodine acetone decolourizer: Take 35ml of liquor iodi fortis and dilute with acetone to make it 1lt. No hold time.
q. Weak-Iodine acetone decolourizer: Take 3.5ml of liquor iodi fortis and dilute with acetone to make it 1lt. 10 second hold time. r. Muir’s Mordant: Mix 20 ml saturated aqueous solution (~7%) of mercuric chloride (HgCl2), 50 ml of saturated (~12%) aqueous potash alum and 20 ml of 20% aqueous Tannic acid and store at RT. s. Plimmer’s mordant: Triturate to dissolve 20 g tannic acid, 36 g of AlCl 3.6H2O and 3 g Basic fuchsin in 80 ml of 60% ethanol. For use dilute 1 part with 3 parts of water. t. Ryu’s Mordant: Dissolve 10 g of tannic acid in 50 ml of 5% aqueous phenol and 50 ml of saturated potash alum.
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u. Rhodes’ mordant: Add 30 ml of saturated aqueous potash alum (~12%) in to 60 ml of 10% aqueous tannic acid solution and then add 6 ml of 3.5% aqueous aniline dye solution, shake the mixture till all precipitate re-dissolves. Add 6 ml of 5% aqueous ferric chloride which yields a black solution. v. Neisser’s staining solution: Mix 20 ml of solution A with 10 ml of solution B.
Solution A: Dissolve 100 mg of methylene blue in 100 ml water and then add 5 ml each of glacial acetic acid and 95% ethanol. Solution B: Dissolve 330 mg of crystal violet in 3.3 ml of 95% ethanol and make it 100 ml with distilled water. w. Ryu’s flagella stain: Mix 10 parts of solution A with one part of solution B and store at RT, allow standing for 2-3 days for use. Freshly made stain is more potent and staining for 5 min is sufficient while for stabilized stain 10 min are required. Solution A (mordant): Dissolve 10 g of tannic acid in 50 ml of 5% aqueous phenol and 50 ml of saturated potash alum. Solution B: Dissolve 12 g of crystal violet in 100 ml of absolute ethanol.
x. Sudan black: Dissolve 300 mg of powdered Sudan Black B in 100 ml of 70% ethanol; allow standing overnight before use in well stoppered bottles. y. Buffers for dilution of Leishman’s stain and washing of slides: Na2HPO4 (anhydrous), 5.447; KH2PO4, 4.752 g are grounded together to homogeneity. Take one gram of buffer and dissolve in 2 lt of distilled water to give Ph of 7.0. To prepare buffer with pH 6.8, take Na2HPO4 (anhydrous), 4.539 and KH2PO4 5.940 g.
38. STAINING METHODS IN MICROBIOLOGY a. Simple staining: Loeffler’s methylene blue or polychrome methylene blue is the best simple stain which reveals many details, beading, barring, protoplasmic granules (Corynebacterium) and for sporulating and capsulating microbes. Stain the heat fixed smear for 1 min- Rinse with water- and drain away the water, blot to dry. b. Gram’s Staining:
62
Lillie’s methods: To heat fixed smear apply ammonium oxalate crystal violet stain for 30
i.
seconds-Wash with water-Apply Lugol’s iodine for 30 seconds-drain off iodine solutiondecolourize with a few drops of acetone for not more than 2-3 seconds-Wash thoroughly with water-apply 0.5% safranin or with weak Carbol fuchsin for 30 seconds-Wash and allow to dry or blot dry.
ii.
Preston and Morrell’s method: To heat fixed smear apply ammonium oxalate crystal violet stain for 30 seconds-Wash with Lugol’s iodine and apply Lugol’s iodine for 30 seconds-wash off with acetone-iodine solution and apply the same for 30 seconds-wash thoroughly with water-apply weak Carbol fuchsin for 30 seconds-Wash and allow to dry or blot dry. For counter staining Carbol fuchsin is preferred by some over Safranin particularly for Yersinia and Haemophillus strains. Bismarck brown is also a preferred counter stain.
iii.
Quick Gram Staining Method: Hold the smear slide with forceps. Flood the slide with methyl violet or crystal violet for 5 seconds and then tip off the stain. Flood the slide with iodine solution and allow to act for 5 second. Tip off the iodine and flood the slide with acetone to act for only 2 seconds. Wash the slide with running water and then flood the slide with basic fuchsin for 5 seconds to counter stain, wash with water, blot and dry.
c. Acid-fast staining (Ziehl Neelsen’s method): Flood the heat fixed slide with strong Carbol Fuchsin and warm to steam but not to boil- re-warm after 3-4 min and leave for 5-7 min- wash off with running water, de-stain with acid-alcohol (3% v/v, HCl in methylated spirit) or 12.5 to 20% sulphuric acid (for 20% solution add 250 ml of concentrated sulphuric acid in 1 lt of distilled water) till all stain goes off with intermittent washing of water- counter-stain with Loeffler’s methylene blue or 0.1 to 0.5% malachite green for 1 min- wash and stand to dry, do not blot. Acid-fast bacteria appear red while others take blue or green stain. For staining the section, clear the section of the paraffin with xylene and alcohol. Then stain as for smear. For weakly acid-fast bacteria de-staining is done 5% sulphuric acid or 1% acid alcohol, instead of 20% sulphuric acid or 1% acid alcohol.
d. Auramine-phenol stain for fluorescence method: Flood the slide with auramine-phenol (dissolve 3 g Auramine O in warm 3% w/v aqueous phenol, filter and store in dark in air-tight bottles) for 10 min, and then wash well under tap water. Decolorize with acid-alcohol for 5 min,
63
wash with water and counter-stain with potassium permanganate for 30 s. Examine slide with fluorescence microscope using blue PY filter in condenser (to cut all short wave light) and yellow filter in eyepiece
e. Spore staining: i.
Moeller’s method: Flood the heat fixed slide with strong Carbol Fuchsin and warm to steam but not to boil- leave for 5-7 min- wash off with running water-de-stain with ethanol till all stain goes off - wash off with water- counter-stain with Loeffler’s methylene blue or 0.5% malachite green for two min- wash and stand to dry. Bacterial bodies stain blue and spores take red colour.
ii.
Schaeffer and Fulton’s method: Flood the slide with 5% malachite green and steam for 1 min- wash off with running water- counter-stain with 0.5% safranin for 15 seconds- wash and allow drying or blotting dry. Bacterial bodies stain red and spores take green colour.
iii.
A modified Ziehl-Neelsen staining procedure: Method is same as for acid-fast bacilli but in this method weak (0.25%) sulphuric acid is used as decolourizer. Procedure yields red spores and blue stained bacteria. Lipid granules also stained red, looking like round spores.
f. Capsule staining: Wet film methods are superior to dry film methods. i.
Muir’s Method: Flood the slide with 5% malachite green and steam for 1 min- rinse quickly with ethanol and wash off with running water- flood the slide with Muir’s mordant for 30 seconds-was off with water and then with ethanol for 30 seconds- wash again with water-counter stain with Loeffler’s methylene blue for 30 seconds- wash and allow to dry or blot dry. Bacterial bodies stain red and capsule take blue colour.
ii.
Giemsa method: Fix the air dried smear with methanol for 3 min or with 1:1000 mercuric chloride- drain off the fixative- flood the slide with Giemsa stain- wash off with 10 mM PBS pH 7.0 for 30 seconds- blot dry. Bacterial bodies appear blue purple and surrounding capsule takes pink-red colour.
iii.
India-ink method (negative staining): Place a loopful of India ink on slide-mix small portion of bacterial colony or pellet from broth culture- put cover-slip and press it with pad of blotting paper to have as thin film as possible of stain (equal to thickness of bacteria, excessive pressing may deform the capsule).
64
g. Staining of lipid granules: i.
Holbrook and Anderson method: Stain the heat fixed smear with 5% malachite green for 2 min over steam (hold slide over boiling water). Wash with water, blot dry. Stain with 0.3% Sudan black B in 70% ethanol for 15 min. Wash film wit xylene for 5 seconds, blot dry. Counter stain wit 0.5% safranine for 20 seconds. Was wit water and blot dry. Lipid granules appear black, spores green and cytoplasm red.
ii.
Burdon’s method: Cover the hat fixed smear with Sudan black stain (0.3 g Sudan black B in 100 ml 70% ethanol) and leave at room temperature for 15 min., drain off excess stain, blot, dry in air. Rinse with xyline, blot dry and counter-stain with 0.5% aqueous safranine or dilute carbol fuchsin for 5-10 sconds. Rinse with water, blot and dry. Lipid granules appear blue-black or blue-grey in light pink bacterial cytoplasm.
h. Metachromatic granule (Volutin granules) staining: i.
Albert’s method: Stain the smear for 2-3 min with Albert’s stain- wash off with water and blot to dry- flood with Albrt’s iodine for 1 min- wash with water and blot dry. Cytoplasm appear greenish and granules blue black.
ii.
Neisser’s method: Stain the smear for 10 seconds Neisser’s stain- wash off with water flood with 0.2% Bismarck brown or 0.4% chrysodin iodine for half min- wash with water and blot dry. Cytoplasm appears light brown and granules blue black.
i. Flagella Staining: For staining, put two drops of distilled water on grease free clean slide- pick a small portion of colony with straight wire- dip the wire tip holding bacteria in each of the two drops just to transfer a few bacteria- allow to dry and then stain with any of the two methods.
i.
Ryu’s method: Flood the slide with Ryu’s stain for 15 min-wash with running water and stand to dry.
ii.
Cesares-Gill’s method: flood the slide with Krickpatric’s fixative for 5 min- wash off with water- flood the slide with filtered and diluted Plimmer’s mordant for 5 min- wash off with water- counterstain with weak Carbol fuchsin- wash with water and stand to dry.
65
iii.
Rhodes’ method: Flood the slide with Rhodes’ mordant for 5 min- wash off with waterapply steaming hot ammonical silver nitrate and leave for 5 min- wash off with water and drain to dry.
j. Fungal staining (for cell polysaccharides): Most fungi are relatively large (>2 μm) and often need not to stain.
i.
Direct microscopy: This technique is used for skin scrapings, sputum, fluid samples and nail-clippings. The test material is mixed with a few drops of 10% aqueous KOH and examined under microscope with subdued light by racking down the condenser.
ii.
Parker-blue staining: Same as direct microscopy, instead of 10% KOH 1:1 mixture of 30% KOH and Parker Blue-black ink is used to soften the preparation and mycelia takes stain. Malassezia furfur can easily be demonstrated in cases of pityriasis versicolor.
iii.
PAS (per-iodic acid Schiff) staining: Although fungi are gram positive but reaction is often non-differentiating and PAS method is preferred. Heat fixed smear is flooded with 0.5% per-iodic acid for 5 min then washed and put in Coleman’s Feulgen reagent for 15-20 min, wash with running tap water for 10 min, fungi stain pink.
k. Staining for Spirochetes: i.
Fontana’s method for films: Treat the film with fixative (Acetic acid 1ml, formalin 2ml and distilled water to make it 100 ml) three times for 30 seconds each. Wash off the fixative with absolute alcohol for 3 min. Drain off alcohol and burn the remaining to dry the film. Apply mordant (phenol 1 g, tannic acid 5 g in 100 ml distilled water), heat to steam for 30 seconds and then wash off with distilled water, dry the slide. Apply ammoniated silver nitrate (add 10% ammonia in 0.5% solution of silver nitrate in distilled water until the precipitate formed get just dissolved, then add more silver nitrate drop wise to reappear precipitate and that does not re-dissolve), heat to steam for 30 seconds and then wash off with distilled water, dry the slide and mount in Canada balsam under a cover-slip. Spirochetes stain brownish black in brownish yellow background.
ii.
Levaditi’s method for staining spirochetes in tissues: Tissue fixed in formalin (10%) for 24 h is washed for 1 h in water and then in 96-98% alcohol for 24 h. Place tissue in 1% silver nitrate added with 10% (final concentration) of pyridine for 2 hr at RT and then at 50 oC for 4-6 h.
66
Rapidly wash the tissue with 10% pyridine and transfer to 4% formalin (in 100 parts of which 10 parts acetone and 15 parts pyridine are added just before use), keep for 2 days. Then wash and dehydrate the tissue and embed the tissue to cut section. Sections are cleared off paraffin and xylene and mounted immediately in Canada balsam.
39. DNA STAINING Acridine orange has good affinity to DNA and RNA and result in to yellow-green and orangered fluorescence under UV light and technique is used to reveal scanty bacteria in wet films of blood cultures. In the process, take living cells, tissue or exudates over cover-slip and without permitting to dry put it into acid alcohol (3% HCl in 95% ethanol) for 5 min. Rinse it with citrate phosphate buffer [pH 3.8, mix 32.3 ml of 0.1 M citric acid (19.21 g/lt) with 17.7 ml of 0.2 M Na2HPO4 (28.39 g/lt) and make the volume 100 ml with distilled water, it is stable only for 2 weeks]. Stain the preparation with 0.01% acridine orange (solution is stable only for 1-2 week in dark) for 4-10 min. Wash twice with buffer for 2 min each. Mount cover-slip over a small drop of buffer and seal the edges. For bacteria, transfer a loopful (1 µl) of culture into an equal size drop of acridine on glass slide. Ring the drop with a ‘wall’ of soft petroleum jelly and apply cover-slip. Examine under UV microscope.
40. FIXATIVES USED IN MICROBIOLOGY: a. Formalin 38-40% formaldehyde (w/v) in water containing 10% methanol (to inhibit polymerization) is used as 10% (4% formaldehyde) in 0.85% NaCl aqueous solution. It tends to be acidic on storage due to formation of formic acid on oxidation of formaldehyde. b. Susa’s fixative: This is one of the best fixatives but tissues should not be thicker than 1 cm and 3-24 h is must to act. And tissues are directly transferred to 95% ethanol (made brown by adding saturated iodine solution) while processing. It contains: Mercuric chloride 45 g, Sodium chloride 5 g, tri-chloro-acetic acid 20 g, acetic acid 40 ml in 800 ml distilled water then 200 ml of formalin is added to make it 1 lt. c. Bouin’s fluid: Good for viral inclusion bodies in tissues or cells. It is effective for tissues less than 1 cm thick and fixes in 1-12 h and tissues are directly transferred to 50% alcohol and then to 70% alcohol until the yellow colour of picric acid is removed. It consists of 75 parts of saturated aqueous picric acid, 25 parts of formalin and 5 parts of glacial acetic acid.
67
d. Schaudinn’s fluid: It is good for protozoa and wet films, can be use cold or warm (60 oC). it consists 1 part of absolute alcohol and 2 parts of saturated aqueous mercuric chloride. e. Flemming’s fluid: Osmic and chromic acid mixture keeps good for 3-4 weeks but acetic acid should be added just before use. It contains 0.1 g osmic acid, 0.2 g chromic acid and 0.1 ml glacial acetic acid in water.
41. GENERAL PROTOCOL FOR EMBEDDING THE FIXED TISSUES: 1.
Small pieces of tissues are transferred to 50% ethanol.
2.
Transfer tissues to 90% ethanol for 2-5 h.
3.
Transfer to absolute alcohol for 2 h.
4.
Repeat step 3.
5.
Transfer to 1:1 alcohol: chloroform mixture to keep overnight.
6.
Place in pure chloroform for 6 h.
7.
Transfer to 1:1 mixture of chloroform: paraffin wax for 1 h at 55oC.
8.
Place in pure melted paraffin at 55oC for two hr.
9.
Tissues are transferred to embedding blocks with molten paraffin.
10.
Sections are cut at 5 µm thicknesses.
42. INHIBITORS FOR SWARMING Swarming is a big problem in isolation of single colony. For inhibition of swarming of Proteus following methods can be used: Agar >2 (2.5 or 3) %, 50 mcg/ml sulphonamides, absence of electrolytes, Bile 1%, PNPG (Para-nitro-phenyl glycerol, 50 mg/l (0.2-0.4 mM for 24 h incubation and 1 mM for 3 day incubation), it can be added in to blood agar also without affecting RBCs and also in to antibiotic sensitivity media).
43.
STERILIZATION: The process, which entirely eliminates viable microbes from a
material or medium. Methods of Sterilization: A. Heat (kills microbes by denaturation and coagulation of proteins). i.
Moist heat (requires lower temperature than dry heat, one of the most economical, safe and reliable method of sterilization). It effectively sterilize at 121-124oC in 15 min and at 134-138oC in 3 min.
68
ii.
Dry heat: requires longer time, higher temperature (oven) i.e. 120 minutes at 160OC or 30 min at 180oC.
Moist heat-Pressure temperature relationship in Autoclave Pressure in pounds
Temperature in OC
Temperature in OF
5
109
228
10
115
240
15
121 (121-124)
250
20
126
259
25
130
267
30
135 (134-138)
275
B. Chemical: Gaseous-formaldehyde (low temperature steam formaldehyde, LTSF, saturated steam at 363 mmHg pressure has a temperature of 73 oC), ethylene oxide and Bpropiolactone, Liquids-gluteraldehyde, hydrogen peroxide (in gas plasma state), per-acetic acid, chlorine dioxide and formaldehyde. C. Radiation: a. Ultraviolet (240 to 260 nm) light is bactericidal produces lethal mutations, stops reproduction, b. Ionizing radiations e.g. gamma rays (usually generated by cobalt-60 (Co60) or other radioactive isotopes), c. High energy X-rays and high energy electrons (ionize the atoms to produce active ions and free radicals which reacts indirectly with DNA.
D. Filtration (excludes microbes rather than killing). For removing bacteria we need filter with 0.2 μm pore size. For removing viruses filter with 0.01-0.1 μm pore size. Analytical filters with pore size of 0.45 μm remove only large bacteria.
Sterility assurance or to determine degree of sterility we use different indicator microbe for different methods: Sterilization method
Indicator Microbe
Steam
Bacillus stearotherophilus (NCTC 10003)
Dry heat
Bacillus subtilis var niger
Formalin
Bacillus stearotherophilus (NCTC 10003)
Ethylene oxide
Bacillus subtilis var globigii or var niger
69
(NCTC10073 or ATCC 9372) Ionizing radiation
Bacillus pumilus
Filtration
Pseudomonas diminuta for 0.22 µm filters and E. coli for 0.45 µm filters.
UV rays
Aspergillus niger spores
44. MEDIA USED FOR STERILITY TESTING: AC broth, AC medium, Agar medium No. F, Brewers thioglycollate medium, Fluid thioglycollate medium, Fluid thioglycollate medium with beef extract, Fluid thioglycollate medium with K agar, NIH thioglycollate medium, tryptic Soy broth, Septum fluid A, Septum fluid D, Septum Fluid thioglycollate medium, Septum tryptic soy broth, thioglycollate medium without dextrose, thioglycollate medium without indicator, thioglycollate medium without dextrose and indicator.
45. INDICATOR ORGANISMS: a. Coliforms: Members of Enterobacteriaceae which grow in presence of bile salts and produce acid and gas from lactose within 48 h at 37 oC (old definition). Redefined as ‘Members of Enterobacteriaceae capable of growing at 37oC and normally possess Bgalactocidase. b. Faecal (thermo tolerant) coliforms: Coliforms capable to grow at 44oC. c. Faecal E. coli: Essential indicator of faecal pollution of human or animal origin. These are defined as thermotolerant coliforms, which ferment lactose (or mannitol) at 44oC and produce indole from tryptophan. d. Faecal Streptococci: These are more resistant to chlorination than coliforms. In human faeces they are less common than E. coli while in animal faeces converse is usually true. These are defined as gram-positive cocci capable to grow at 45oC in presence of 40% bile salts and sodium azide. These can survive at 60oC for 30 min and can grow at 10oC and pH 9.6 and in 6.5% NaCl. Commonest species in human beings are; Enterobacter faecium and E. faecalis. Some faecal cocci those do not have this repertoire include Streptococcus bovis and S. equinus found in faeces of bovines (also in human) and equines, respectively. e. Sulphite reducing clostridia: Members of genus Clostridium reducing sulphate to Sulphide including C. perfringens, spores survive longer and are used as indicator of remote or intermittent faecal pollution.
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f. Pseudomonas: Though not a indicator of pollution but used to monitor water supply in food establishments, hospitals and pharmaceuticals due to its opportunistic pathogen role. g. Bacteriophages: Coli phages are good indicator of faecal pollution and have chlorine resistance similar to human entero-viruses thus may be useful indicator of viral pollution.
46. SAMPLING PLAN FOR POLLUTED WATERS: Type water
of 50 ml in to 50 ml 10 ml in to 10 ml 1 ml in 5 ml 0.1 ml in 5 ml double medium Durham tube
strength double
strength single
with medium Durham tube
strength medium
with medium
with
with Durham tube
Durham tube
Good quality 1 5 Doubtful 1 5 5 quality Known to be 5 5 5 polluted MPN Media used for faecal coliforms are: laural tryptose broth, brilliant green lactose bile broth, laural lactose broth. Positive control is E. coli while K. pneumoniae ssp. aerogenes is negative control. MPN Media used for sulphate reducing clostridia are: Differential reinforced clostridial medium (DRCM).
47. MEMBRANE FILTRATION TEST FOR INDICATOR MICROBES : Sterilized membrane of 0.45 μm are used and vacuum of about 500mmHg is used for filtration of 100 ml volume of test sample. Membranes are aseptically removed from the filtration funnel and laid on to suitable paper pads (≥1.4 mm thick) saturated with a selective indicator membrane medium. For coliforms, membrane laural sulphate broth permits formation of yellow colonies of coliforms. Membranes are incubated at 30 oC for 4 hrs and then at 37oC for 14 hrs. Yellow colonies capable to produce indole, oxidase negative and able to ferment lactose to form acid and gas at 44oC are classified as confirmed coliforms. For Enterococci count Membrane Enterococcus agar is used, enterococci form red, maroon and pale pink colonies, which can be confirmed on bile aesculin azide agar, formation of brown-black colour at 44-45oC in few hrs (`12-18 hrs) is confirmatory. For clostridia membrane clostridial agar is used for layering over membrane. For drinking water there should be no coliforms, faecal coliforms and faecal streptococci in 100 ml sample and not more than 1 sulphite reducing clostridia. There should no not more than 100 and 10 colonies, in 1 ml water, on incubation at 22 and 35oC, respectively.
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48. AIR SAMPLING A. Settle plate method: Petri dishes containing an agar medium of known surface area are left open for a measured period of time. Bacteria containing particles settle on the medium and form colonies on incubation. Blood agar is used overall count of bacteria in air; specific selective media can be used for specific bacteria/ fungi. Generally aerobic incubation at 37 oC for 24 hr is for most pathogens but for saprophytes and moulds it is 3 days at 22 oC and 1-2 weeks at 22oC, respectively. Exposure of plates should be usually at a height of 1 m of flat surface. Disadvantage of the method is that it only determines the settling rate of large particles and not the total number of bacteria present on all kinds of particles in air.
B. Slit sampler: It is the most convenient and efficient device for counting bacteria carrying particles in unit volume of air. Device draws air at a fixed rate (1-20 cubic foot/ min, for ultra clean air it draws air at 700 litres /min rate) and causes the suspended particle to settle on surface of agar medium. It captures bacteria in aerosol and even those particles, which are less than 0.25 micron in size. In it an air tight box connected with a vacuum pump having a slowly rotating platform to hold the media plate. The slit (0.33 mm wide, 27.5 mm long and 3 mm deep) is positioned just 2 mm above the surface of agar. Incoming particles get deposited on agar plate and rotation of plate permit to settle them on all surface of agar than getting localized. Level of vacuum in box determines the rate of incoming air. Bacterial contamination of air is expressed as Bioload (B) unit [(bacteria-carrying particles per cubic meter (bcpm-3)]. In normal air
