This labtop monograph is developed for new as well as experienced microbiologists to help them in finding all the day to day information at one place to minimize the labour of searching the needed information from pages of voluminous editions of microbiology books. The monograph encompasses information on procedures (standard as well as short cuts) for biochemical tests, making buffers, antibiotic solutions, media and dye solutions (indicator solutions) for characterization of bacteria through conventional and modern methods. Conventional and special staining procedures, sterilization methods and preservation of microbes are described in simple language.
• Labtop for Microbiology LaboratoryTable of Contents S No Page number 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 Buffers/solutions for SDS-PAGE 13-14 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 13 14 Gel processing solutions: ELISA-reagents and buffers Solutions for estimation of protein 15 15-17 17-18 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 16 laboratory Physical characteristics of common acids and alkalies 19 17 used in laboratory 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 i Borate calcium saline, pH 7.3 20-25 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 m) p Potassium phosphate buffer for pH 5.8 to 8.0 (0 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 B Swarm-agar (Guard plate) 25-26 26 27 28-30 30 31 31-33 33 332 33-35 26 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 Ao Media for antigen extraction from streptococci for grouping 27 28 29 30 31 32 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 C Precipitation with water miscible organic polymers as polyethylene glycol 43-45 45-46 46 47 47 48 33 34 35 36 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 X) v Phosphatase test 48-49 49-50 50-51 52-60 w x y z 37 38 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 iii India-ink method (negative staining) G Staining of lipid granules 60-63 63-67 39 40 41 42 43 44 45 46 47 48 49 50 51 52 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 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 • Labtop Book for Microbiology- 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 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 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 56 126 224 349 503 685 894 1132 1398 1691 2012 2362 2739 3144 3578 4039 4528 5045 5590 6163 6764 7393 8050 9447 10188 10956 RCF with Rotor Radius (from center of rotor to sample) in centimeters 10 11 12 13 67 78 89 101 112 123 134 145 151 176 201 226 252 277 302 327 268 313 358 402 447 492 537 581 419 489 559 629 699 769 839 908 604 704 805 906 1006 1107 1207 1308 822 959 1096 1233 1370 1507 1643 1780 1073 1252 1431 1610 1789 1968 2147 2325 1358 1585 1811 2038 2264 2490 2717 2943 1677 1957 2236 2516 2795 3075 3354 3634 2029 2367 2706 3044 3382 3720 4058 4397 2415 2817 3220 3622 4025 4427 4830 5232 2834 3306 3779 4251 4724 5196 5668 6141 3287 3835 4383 4930 5478 6026 6574 7122 3773 4402 5031 5660 6289 6918 7547 8175 4293 5009 5724 6440 7155 7871 8586 9302 4847 5654 6462 7270 8078 8885 9693 10501 5433 6339 7245 8150 9056 9961 10867 11773 6054 7063 8072 9081 10090 11099 12108 13117 6708 7826 8944 10062 11180 12298 13416 14534 7396 8628 9861 11093 12326 13559 14791 16024 8117 9469 10822 12175 13528 14881 16233 17586 8871 10350 11828 13307 14786 16264 17743 19221 9660 11269 12879 14489 16099 17709 19319 20929 11337 13226 15115 17005 18894 20784 22673 24562 12225 14263 16300 18338 20376 22413 24451 26488 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 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 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 day or more and then cleaned with water 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 ml Normal saline solution (0.85%) 900.0 ml Both solutions were mixed and stored in a capped jar 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 water100 ml 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, g/lt 100 100 100 100 KCl, 11.5 g/lt 4 4 CaCl2, 12.2 g/lt 3 3 KH2PO4, 21.1 g/lt 1 MgSO4.7H2O, 38.2 g/lt 1 NaHCO3, 13.0 g/lt 21 Phosphate buffer, 0.1M, - Phosphate 100 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 Alsever’s Solution Glucose 1.866 g 10 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 30oC for 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 4445oC 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 sulphite reducing clostridia There should no not more than 100 and 10 colonies, in ml water, on incubation at 22 and 35oC, respectively 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 days at 22 oC and 1-2 weeks at 22oC, respectively Exposure of plates should be usually at a height of 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 mm deep) is positioned just mm above the surface of agar Incoming particles get deposited on 70 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 [...]... 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 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... 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... 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... 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... 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... 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 Lactobacilli Agar (Difco) can also be used for maintenance... 1 lt pH 7.6 Never heat above 100oC 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... 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... 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... 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 115 oC for 10 min Falkow modified medium (1958) is made by dissolving 5 g peptone, 3 g yeast extract and 1 g glucose ... 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... 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 • Labtop Book for Microbiology- RATES IN CENTRIFUGATION: a Settling... 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