PRACTICAL MANUAL ON MICROBIOLOGICAL TECHNIQUES III (STM412)HND II

MCB 1ST SEMISTER

EXP 1 DATE..........................
TITTLE:-Preparation of Indicator Solutions
AIMS:-To prepare indicator system
PRINCIPLE:-Preparation of all indicator solution used in chemical analysis of different
pharmaceutical products, their pH range and change in colour at different pH values during analysis.
In the test and assays of the pharmaceutical ingredients, indicators are required to indicate the
completion of a chemical reaction in volumetric analysis or to indicate the pH of solutions. Indicators
may be substituted for one another provided the colours change over approximately the same range of
pH but in the event of doubt or dispute as to the equivalence of indicators for a particular procedure,
the indicator specified in the individual monograph is alone authoritative. Any solvent required in a
determination or test in which an indicator is specified should be previously neutralized to the
indicator unless a blank determination is performed or specified.
METHODOLOGIES
Indicator solution 1: Congo red fibrin indicator
Chemical formula and nomenclature
Congo Red Indicator; CI 22120; Disodium (4,4' -bipheny Ibis- 2,2-azo) bis(1-aminonaphthalene-4-
sulphonate): C 32H 22N 6Na2 O6S 2= 696.66
Properties
Dark red or reddish brown powder.
Decomposes on exposure to acid fumes.
Congo red Fibrin Indicator preparation procedure
.Soak washed and shredded fibrin overnight in a 2 per cent w/v solution of congo red in ethanol (90
per cent), strain, wash the product with water and store under ether.
Indicator solution 2: Crystal violet indicator
Chemical formula and nomenclature
Crystal Violet Indicator; CI 42555; Basic Violet 3; Hexamethylp-rosanilineChloride: C 25H 30CIN
3= 407.98
Properties
When used for titrations in non-aqueous media, changes from violet (basic) through blue-green
(neutral) to yellowish green (acidic).
Crystal Violet Indicator Solution preparation procedure
A 0.5 per cent w/v solution of crystal violet in anhydrous glacial acetic acid. Complies with the
following test.
SENSITIVITY - A mixture of 0.1 ml of the solution and 50 ml of anhydrous glacial acetic acid is
bluish purple. Add 0.1 ml of 0.1 M perchloric acid; the solution turns blue-green.

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Indicator solution 3: Methyl red indicator
Chemical formula and nomenclature
Methyl Orange Indicator; CI 13025; Sodium 4-dimethylaminoazobenzene- 4-sulphonate: C 14H 14N
3NaO 3S = 327.34
Preparation Procedure
Dissolve 0.1 g of methyl orange in 80 ml of water and add sufficient ethanol (95 per cent) to produce
l00 ml. Complies with the following test.
SENSITIVITY - A mixture of 0.1 m] of the solution and 100 ml of carbon dioxide-free water is
yellow. Not more than 0.1 ml of 0.1 M hydrochloric acid is requiredto change the colour to red.
EXP2 DATE........................
TITTLE :-Anaerobic Bacteriology
AIMS:-CULTIVATION OF ANAEROBIC  BACTERIA 
INTRODUCTION:One of the environmental factors to which bacteria and other microorganisms are
quite sensitive is the presence of O2. For example, some microorganisms will grow only in the presen
ce of O2 and are called obligate aerobes. Facultative anaerobes will grow either aerobically or in the a
bsence of O2, but better in its presence. Strict obligate anaerobes will grow only in the absence of O2 
and are actually harmed by its presence. Aerotolerant anaerobes are microorganisms that cannot use O
2 but are not harmed by it either. Finally, microorganisms that require a small amount of O2 for norm
al growth but are inhibited by O2 at normal atmospheric tension are called microaerophiles. These var
iations in O2 requirements can be easily seen by inoculating a tube of molten agar with the bacterium 
in question, mixing the agar thoroughly without aerating it, and allowing it to solidify. The bacteria wi
ll grow in the part of the agar deep culture that contains the proper O2 concentration The damaging ef
fects of O2 on anaerobic bacteria create difficult culturing problems. Ideally, one should not only prov
ide an O2‐free environment, but one that has an adequate amount of moisture for bacterial growth. It i
s also necessary to have CO2 present 
for the growth of many anaerobic bacteria. There are a number of ways in which anaerobic bacteria m
ay be cultured. For those bacteria that are not really fastidious anaerobes, growth can occur on nutrien
t agar slants if anaerobic conditions are created. This setup is called a Wright’s tube (named after Jam
es H. Wright, American physician, 1901–1978) 
The anaerobic condition is created using pyrogallol and NaOH In the presence of NaOH; pyrogallol is 
oxidized and removes O2 very effectively in the process. After the anaerobic bacterium has been strea
ked out on the surface of the agar slant, the cotton plug is trimmed and then pushed into the culture tu
be  unt
il i t re
sts  jus
t a bo
ve  th
e t op 
of t he 
sla nt. 

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The space between the top of the cotton plug and the open end of the culture tube is then filled with p
yrogallol cr
ystals, and 
1 ml of 10
% NaOH is 
added. The 
tube is clos
ed with a ru
bber stoppe
r and is im
mediately i
nverted. It i
s incubated 
upside dow
n. Anaerobi
c bacteria m
ay also be g
rown in spe
cial anaerob
ic  jar syste
m called Ga
sPak Anaer
obic Syste
m. In the G
asPak Syste
m
figure 3: or
in The
Candle jar
as in figure
2:

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Hydrogen and CO2 are generated by a GasPak envelope after the addition of water. A palladium catal
yst (pellets) in the chamber lid catalyzes the formation of water from hydrogen and O2, thereby remo
ving O2 from the sealed chambr.
Figure1: Preparation of an Anaerobic Wright’s Tube. Pyrogallol is a reducing agent that is activated
by NaOH to remove oxygen from the tube and create anaerobic conditions.  

PRINCIPLES: One of the most convenient approaches to create anaerobiosis is is to employ a specia
lly designed commercial anaerobic broth. Two of the most useful are cooked meat medium and thiogl
ycollate broth. Thioglycollate medium can be purchased with methylene blue or resazurin as an oxidat
ionreduction indicator. When this medium begins to turn bluish or 
reddish, it is becoming too aerobic for the culture of anaerobic bacteria. 

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Materials 
‐24‐ to 48‐hour broth cultures of Pseudomonas aeruginosa, Escherichia coli  and ‐Clostridium sporoge
nes  ‐boiling  water bath  ‐48° to 50°C water bath  ‐4 thioglycollate broth tubes  ‐
inoculating loop  ‐2 tryptic soy agar plates  ‐sterilized Brewer’s anaerobic cover -
GasPak Anaerobic System   ‐4 tryptic soy agar slants  ‐scissors  ‐cotton plugs  ‐
pyrogallol crystals (poisonous)  ‐10% NaOH (caustic)  ‐test tubes  ‐rubber stoppers  ‐test‐
tube rack  ‐wax pencil  ‐disposable gloves  ‐spatula for handling pyrogallol crystals and soil  ‐1‐
ml pipette with pipettor  ‐garden soil 
Method 

The Relationship of O2 to Bacterial Growth 

1. Melt three  tryptic  agar deeps and heat them in a boiling water bath for a few minutes in 
order to drive off any O2. 
2. Cool the deeps in a water bath 48° to 50°C. 
 3. With a wax pencil, label each tube with the name of the bacterium to be inoculated, your 
name, and date. 
4. Using aseptic technique inoculate each cooled deep with 1 or 2 loopsful of one of each of 
the three different bacteria (P. aeruginosa, C. sporogenes, and E. coli). 
5. Mix the bacteria throughout the agar without aerating it by rolling each tube between the 
palms of your hands. 
6. Allow the agar to harden and incubate the three tubes for 24 to 48 hours at 35°C. 
Broth Culture of Anaerobic Bacteria 
1. With a wax pencil, label three freshly steamed thioglycollate broth tubes with P. 
aeruginosa, C. sporogenes, and E. coli, as well as your name and date. 
2. Using aseptic procedures, inoculate the three broth tubes. Do not shake these tubes to 
avoid oxidizing the medium! Methylene blue or resazurin is present in the medium as anoxidation‐re 
duction indicator. If more than 1/3  the broth is bluish or reddish in color, the tube should be reheated 
in a water bath in order to drive off the O 2 before use.  

3. Incubate the tubes at 35°C for 24 to 48 hours.. 
Slant Culture of Anaerobic Bacteria in a Wright’s Tube 
1. With a wax pencil, label four tryptic soy agar slants,2 with P. aeruginosa and 2 with 
C. sporogenes, and your name and date. 
2. Using aseptic technique, inoculate each slant with the respective bacterium. 
3. Incubate two slants (one of each bacterium) aerobically at 35°C for 24 to 48 hours. 
4. With the other two slants, cut off the cotton plug  with a pair of scissors (figure 2), and 
with the butt of the inoculating loop, push it into the tube until it almost touches the toplateof the slant
.While  wearing gloves, fill the space above the cotton with pyrogallol crystalsand add 1 ml of 10% N
aOH. Quickly stopper the tube with a rubber stopper and invert the  tube. 

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5. Place in a test‐tube rack and incubate inverted at 35°C for 24 to 48 hours. Isolation of Anaerobes fr
om the Soil 
1. Place a spatula of soil in a thioglycollate broth tube. 
2. Incubate at either 30° or 37°C for one day  
3. Observe culture tubes for growth in the form of turbidity and the production of  fermentation gases.
 B.SOLATION OF ANAEROBIC BACTERIA FROM CLINICAL MATERIAL
Prepare and stain direct smears from each specimen.
In order to gain some insight into the quantity and type of organisms in the specimen, examine a gram
stained smear. Examination of wet mounts of unstained material, acid fast stained smears, and Giemsa
stained smears may also be helpful. Use capillary pipettes to prepare smears from liquid specimens or
use swabs directly.
Observe and record:
1. The gram reaction, size, shape and relative numbers of organisms present.
2. The presence of spores and their shape and position in the cell.
3. Any distinctive morphological features such as branching, pseudo-branching, chaining, filaments,
spherical bodies, or minute granular forms.
C. Inoculate primary isolation media as soon as possible after specimens are received.
Fluid specimens: Use a capillary pipette to inoculate liquid or semisolid media near the bottom with
one to two drops of inoculum. Place one drop on each plating medium and streak for isolation.
Tissue or other solid specimens: Mince with sterile scissors, add sufficient pre-reduced broth to
emulsify the specimen, add sterile sand as necessary, and grind with a mortar and pestle. Treat as a
liquid specimen.
Swabs: Place swab directly into liquid media and use separate swab to streak plates. If necessary, an
inoculating suspension can be prepared by gently scrubbing the inoculum off a swab in approximately
2 ml of prereduced broth.
1. Heat all liquid and semisolid media in a boiling water bath for 10 minutes and cool before
inoculation.
2. Inoculate one tube each of thioglycollate and chopped meat-dextrose medium (for enrichment
cultures) and two blood agar plates. Add 0.5 ml sterile rabbit serum to the thioglycollate medium after
the medium has been heated and cooled. Laked blood agar plates are recommended for the isolation
of Bacteroides melaninogenicus.
a. If clostridia are suspected inoculate one egg yolk agar plate (EYA) or a plate of stiff blood agar (4%
to 6% agar).
b. If mixed bacterial populations are suspected, inoculate selective plating media, such as phenylethyl
alcohol blood agar10 and/or kanamycin-vancomycin-menadione blood agar.11 Paromomycin or
neomycin may be substituted for kanamycin.

3. Incubate cultures at 35-37°C as follows:

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a. Incubate chopped meat and thioglycollate media in an anaerobe jar for 24 - 48 hours. Loosen caps
to allow exchange of gasses.
b. Incubate one BAP in a candle jar to determine aerobic flora.
c. Incubate one BAP, EYA plate, and selective plating media in an anaerobe jar for a minimum of 48
hours, preferably 3 to 5 days. To absorb excess moisture in plates, use glass Petri dishes (100 X 15
mm) with metal covers containing absorbent discs or:
(1) Add 2-3 drops of glycerin to the lid of each plate.
(2) Place a piece of filter paper in the lid.
(3) Replace bottom of plate, pressing the filter paper into the dish top.
D. After 24 hours incubation, examine enrichment cultures. If no growth is apparent, reincubate
cultures.
1. Prepare and examine Gram stained preparations from thioglycollate and cooked meat-dextrose
cultures.
2. Remove inoculum from near the bottom of the culture tubes with a capillary pipette and inoculate
plating media for subculture.
a. Inoculate two blood agar plates from either the thioglycollate or cooked meat-dextrose cultures.
(1) If clostridia are suspected, inoculate an egg yolk agar12 plate or stiff blood agar.
(2) If the culture contains a mixture of organisms, inoculate selective plating media.
b. Incubate plates in a candle jar or anaerobically as outlined in Section C, 3.
3. Reincubate enrichment cultures if organisms seen on direct smear are not present in cultures.
E. After incubation:
1. Examine anaerobic and CO2 plates with a hand lens and a dissecting microscope.
a. Observe and record the action on blood and egg yolk, and the size and shape of colonies.
(1) Prepare Gram stained smears for comparison of colonies on the different plates. Record shape and
location of any spores observed.
(2) Colonies on egg yolk agar may be used to test for catalase by adding a drop of 3% H2O2 to a
suspension of organisms on a slide. Expose the EYA plates to air for at least 30 minutes before testing
for catalase. Do not use colonies from blood agar plates to test for catalase.
b. Determine the number of different colony types on the anaerobe plates.
(1) For each colony to be transferred, prereduce one tube of chopped meat-dextrose medium and one
tube of thioglycollate medium by heating the media in a boiling water bath for 10 minutes. Cool
before use.
(2) Using a needle with a small loop or a heat sealed 9" Pasteur capillary pipette, fish each different
colony and inoculate a tube of chopped meat-dextrose medium and a tube of thioglycollate medium.
If anaerobes other than clostridia are suspected, add 0.5 ml sterile rabbit serum to the thioglycollate
medium.
Chopped meat medium is best for culturing the clostridia and the enriched thioglycollate medium is
more suitable for the nonsporeforming anaerobes.

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c. Incubate chopped meat and thioglycollate media in an anaerobe jar for 24 - 48 hours.
2. Be sure to have at least one representative colony of each morphological type seen on the original
smear. If necessary, restreak plating media to obtain isolated colonies.
F. Examine plates inoculated with enrichment cultures after incubation. Subculture any colony types
not isolated from direct plates to prereduced chopped meat-dextrose and thioglycollate media.
G. Examine thioglycollate and chopped meat subcultures from isolated colonies. If pure, use these
cultures to inoculate appropriate differential media for identification of the isolates.
TABLE III
Clinical Isolates of Anaerobic Bacteria Most Frequently identify Anaerobinpc Bacteriology Unit
1. Clostridia
C. bifermentam
C. butyricum
C. cadaveris (C. capitovale)*
C. innocuum
C. limosum (Clostridium sp. CDC group P-l)
C. perfringens
C. ramosum (Catenabacterium filamentosum, Bacteroides terebrans)
C. septicum
C. sordellii
C. sporogenes
C. subterminale
C. tertium
2. Nonsporeforming gram-positive bacilli
Actinomyces israelii Actinomyces odontolyticus Actinomyces naeslundii Arachnia propionica
(Actinomyces propionicus) Bifidobacterium eriksonii (Actinomyces eriksonii) Eubacterium
alactolyticum (Ramibacterium species) Eubacterium lentum (Corynebacterium diptheroides)
Eubacterium limosum
Propionibacterium acnes (Corynebacterium acnes) Propionibacterium granulosum (Corynebacterium
granulosum)
3. Nonsporeforming, gram-negative bacilli
Bacteroides fragilis ssp.fragilis (B. fragilis)
Bacteroides fragilis ssp. thetaiotaomicron (B. variabilis) Bacteroides fragilis ssp. vulgatus (B.
incommunis) Bacteroides melaninogenicus ssp. asaccharolyticus Bacteroides melaninogenicus ssp.
intermedius Fusobacterium mortiferum (Splmerophorus ridiculosum) Fusobacterium necrophorum
(Sphaerophorus necrophorus) Fusobacterium nucleatum (Fuosbacterium fusiforme)
4. Anaerobic cocci

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Peptococcus sp. group 2 Peptostreptococcus sp. group 1 Peptostreptococcus sp. group 2
Peptostreptococcus sp. group 3 Veillonella alcalescens Veillonella parvula
Result : Record your growth results 
Question : (1) Explain how an anaerobic atmosphere can be created in a jar. 
(2)Explain what happens in a Wright’s tube. (3) Differentiate between the following: (a)an obligate an
aerobe(b) an obligate aerobe  (c) a facultative anaerobe  (d) an aerotolerant anaerobe 
(e) a microaerophile 
EXP 3 DATE....................
TITTLE :Reed and muench assay
AIMS:-To Measure viruses by end-point dilution assay(Reed and muench)
INTRODUCTION:-The plaque assay is a terrific method for determining virus titers, but itdoesn’t
work for all viruses. Fortunately there are several alternativemethods available, including the end-
point dilution assay.The end-point dilution assay was used to measure virus titer before
thedevelopment of the plaque assay, and is still used for viruses that do notform plaques. Serial
dilutions of a virus stock are prepared andinoculated onto replicate cell cultures, often in multi-well
formats (e.g.96 well plastic plates). The number of cell cultures that are infected isthen determined for
each virus dilution, usually by looking for cytopathiceffect.In this example of an end-point dilution
assay, 10 monolayer cell cultureswere infected with each virus dilution. After an incubation period,
platesthat displayed cytopathic effects were scored with a +. At high dilutions,none of the cell cultures
are infected because no particles are present.At low dilutions, every cell culture is infected. Half of the
cell culturesshowed cytopathic effects at the 10-5 dilution. This is the end point: thedilution of virus at
which 50% of the cell cultures are infected. Thisnumber can be calculated from the data and
expressed as 50%infectious dose (ID 50) per milliliter. The virus stock in this examplecontains 105
ID50 per ml.In real life, the 50% end point does not usually fall exactly on a dilutionas shown in the
example. Therefore statistical procedures are used tocalculate the end point of the titration.End-point
dilution methods can also be used to determine the virulenceof a virus in animals. The same approach
is used: serial dilutions ofviruses are made and inoculated into multiple test animals. Infection ofthe
animal can be determined by death or clinical symptoms such asfever, weight loss, or paralysis. The
results are expressed as 50% lethaldose (LD 50 ) per ml or 50% paralytic dose (PD 50 ) per ml when
lethality orparalysis are used as end points.The following example illustrates the use of end point
dilution tomeasure the lethality of poliovirus in mice. Eight mice were inoculatedper virus dilution,
and the end point was death.
PRINCIPLES :-The statistical method ofReed and Muench was used to determine the 50% end
point. In thismethod, the results are pooled, and the mortality at each dilution iscalculated. The 50%
end point, which falls between the fifth and sixthdilutions, is calculated to be 10 -6.5 . Therefore the
virus sample contains106.5 LD50 units.The Reed–Muench method is a simple method for
determining 50% endpoints inexperimental biology, that is, the concentration of a test substance
thatproduces an effect of interest in half of the test units. Examples include LD50(the median lethal
dose of a toxin or pathogen), EC50 and IC50 (half maximaleffective or inhibitory concentration,
respectively, of a drug), and TCID50 (50%tissue culture infectious dose of a virus).The reason for
using 50% endpoints is that many dose-response relationships inbiology follow a logistic function that
flattens out as it approaches the minimaland maximal responses, so it is easier to measure the
concentration of the testsubstance that produces a 50% response.
PROCEDURES

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1. Dilute log-phase Sf9 cells (with greater than 98% viability) to 1 x 10 5 cells /mlwith fresh TNM-
FH medium. Seed 1 x 10 5 Sf9 cells per well on a 12-well plate(BD Falcon™, Cat. No. 353043).
Allow cells to attach firmly, approximately 10min. Confirm 30% confluency by visualization on a
lightmicroscope. Replacemedium with 1 ml fresh TNM-FH.
2. Add 100, 10, 1 and 0 µl of the recombinant virus supernatant obtained 5 daysafter the start of co-
transfection (or other virus stock), to separate wells. Do thesame for the positive control, e.g.,
pVL1392-XylE supernatant.
3. Incubate the cells at 27°C for three days. Examine the cells for signs ofinfection.
4. A successful transfection should result in uniformly large infected cells in the100, 10, and 1 µl
experimental wells. The cells in the 0 µl control wells should notlook infected because they were not
inoculated with virus.
5. If only the 100 µl and 10 µl wells seem to have infected cells and the 1 µl welllooks more like the
control, the titer of your virus supernatant is low. Amplify thevirus an additional time before
proceeding with protein production.Protein production can be analyzed by western blot analysis (if
antibodies areavailable) or by Compasses blue-stained SDS-PAGE gel by harvesting cells fromthe
100 µl well and lysing in appropriate lysing buffer. The virus supernatant from the 100 µl well may be
kept as the first viralamplification stock, however care should be taken to avoid cross-
contaminationbetween wells containing different virus. To further purify the virus population, a
plaque assay purification of the co-transfection supernatant may be performed using the approximate
titer obtainedfrom the EPDA.
CALCULATION
Reed and Muench method
log 50% end point dilution = log of dilution showing a mortalitynext above 50% - (difference of
logarithms × logarithm of dilutionfactor).
Generally, the following formula is used to calculate “difference oflogarithms” (difference of
logarithms is also known as “proportionate distance” or “interpolated value”): Difference
oflogarithms = [(mortality at dilution next above 50%)-50%]/[(mortality next above 50%)-(mortality
next below 50%)].
Spearman-Karber method
log 50% end point dilution = - (x - d/2 + d ∑ r /n )
x = log of the reciprocal of the highest dilution (lowest concentration) at which all animals are
positive;
d = log of the dilution factor;
n = number of animals used in each individual dilution (afterdiscounting accidental deaths);
r = number of positive animals (out of n ).Summation is started at dilution x .

Newly proposed method

Formula 1: log 50% end point dilution = -[(total number of animals died/number of animals
inoculated per dilution) + 0.5] × log dilutionfactor.

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Formula 2 (if any accidental death occurred): log 50% end point dilution = -(total death score + 0.5)
× log dilution factor.
EXERCISE : (1)Mention the advantages and disadvantages of reed and muench assay. (2)State the
application of the test. ( 3)explain LD50
EXP 4 DATE............................
TITTLE:-Preparation of antibiotic stock solutions and driedpaper discs
AIMS:-To Prepare antibiotic stock solutions and dried paper discs
INTRODUCTION:-Antibitiotics may be received as powders or tablets. It is recommended toobtain
pure antibiotics from commercial sources, and not use injectablesolutions.Powders must be accurately
weighed and dissolved in the appropriate diluentsto yield the required concentration, using sterile
glassware.Standard strains of stock cultures should be used to evaluate the antibiotic stock solution. If
satisfactory, the stock can be aliquoted in 5 ml volumes andfrozen at -20ºC or -60ºC.
MATERIALS
Weighing balance, antibiotics powder/tablet, whattsman filter paper no1, distilled water/diligent,
scissors /blades, micropippette, Petridishes, hot air oven
PROCEDURE
A. Stock solutions are prepared using the formula (1000/P) * V * C=W, where
1. P= potency of the antibiotic base,
2. V=volume in ml required,
3. C=final concentration of solution and
4. W=weight of the antimicrobial to be dissolved in V.
B. Preparation of dried filter paper discs
.Whitman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are
placed in a Petri dish and sterilized in a hot air oven.
.Dispense 0.005 ml (5 microliter) of antibiotic solultion using sterile micropipette tips.
.allow to air dry and
.To test it impregnate the discs on to a fresh media to be tested and and incubate at 27c/24hrs.
EXERCISE: (1) explain potency with regard to antibiotics. (2) what do you understand by stock
solution.

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