Bacteriologic diagnosis can also be confirmed by
estimating antibodies to specific antigens of the bacteria.
Examples: VDRL and Kahn tests for syphilis. ASO for
β-hemolytic streptococci and Widal for typhoid.
Anything used for preparing culture media should be
free from living organisms. All media prepared should
be sterilized according to instructions for each type of
media. pH adjustment should be correct for all media.
Since, most organisms grow at a slightly alkaline pH, it
should therefore be adjusted between pH 7.2–7.6. Time
can be saved by using dehydrated culture media: as per
the manufacturer’s instructions, weigh the dehydrated
medium, add the requisite amount of boiled distilled
water, mix the two and sterilize the solution. Given below
are methods for preparation of culture media:
This medium is used for the testing of indole production,
for the preparation of sugar media, and when made highly
alkaline (pH 8.0–8.4) is used for the cultivation of Vibrio
Dissolve by steaming. Adjust the pH to 7.5. Filter through
paper. Distribute in tubes or bottles. Sterilize at 15 lb
pressure for 20 minutes. The commercially available
peptone water consists of water-soluble products obtained
from lean meat or other protein materials by digestion
mainly with a proteolytic enzyme like pepsin, trypsin
or papain. The important constituents are peptones,
proteoses, amino acids and inorganic salts.
Mix the ingredients and allow to dissolve (can be
accomplished by steaming it). Adjust pH to 7.6. Phosphates
may precipitate out and should be extricated by filtration.
Distribute the medium in large bottles and then sterilize
at 15 lb for 20 minutes. When 1% glucose is added to this
nutrient broth it becomes glucose broth.
Agar-agar is a long chain polysaccharide substance from
certain seaweeds. It forms a firm gel in watery solution at
concentrations of about 2%. Agar alone has no nutritive
properties. It melts at about 95oC and solidifies only when
To the nutrient broth add 2% of agar—it then becomes
nutrient agar. After addition of 2% agar, autoclave at
828 Concise Book of Medical Laboratory Technology: Methods and Interpretations
15 lb for 20 minutes. Clear with white of egg. Autoclave
and filter. Distribute into flasks and sterilize at 15 lb for
Melt the nutrient agar and cool to 50oC. Aseptically add
5–10% sterile defibrinated sheep (ideally) blood. Mix and
pour into petri dishes or tubes which are sloped. Bank
blood or rabbit blood may be used.
Add blood to nutrient agar as for blood agar. Mix well and
raise the temperature to 80oC keeping well mixed. Leave
at 80oC for 10 minutes. Pour into petri dishes or tubes as
Sugar media are used to study the biochemical reactions of
bacteria. To sterilized peptone water add 1% of the required
sugar and 1% Andrade’s indicator. Distribute into sterile tubes
containing inverted Durham’s fermentation tubes. Indicator
is used to study the acid formation by bacteria. If acid is
produced media becomes reddish pink. Instead of Andrades
indicator the following indicators can also be used.
Neutral red 0.25% to 1% solution—if acid is produced—
Phenol red 0.01%—if acid is produced—yellow color.
The sugar media are sterilized by fractional sterilization
or tyndallization. The sugar may be caramelized or charred
at a temperature higher than 100oC, so it is steamed on
three consecutive days in Arnold’s steam sterilizer.
Preparation of Andrade’s Indicator
Dissolve 0.5 gram of acid fuchsin in 100 mL of distilled
water. Add 16 mL of normal sodium hydroxide (NaOH)
and leave overnight. The color should change from pink to
brownish red and then to yellow.
This is used for biochemical reaction of Neisseriae.
Corynebacterium and other organisms requiring serum
Adjust reaction to pH 7.5 and Andrades indicator 1% and
sugar 1%. Sterilize as for peptone water/sugar media.
(Used for cultivating diphtheria bacilli)
Blood is collected from ox under sterile conditions and
serum separated aseptically. Inspissate the medium at
Media for Identification of Fungi
Dissolve peptone in water and adjust the pH to 5.4. Add
agar and melt it at 15 lb. for 20 minutes. Then add glucose
and sterilize by fractional sterilization.
READY TO POUR, STERILIZED POUCHED MEDIA
FOR MICROBIOLOGICAL APPLICATIONS
(Courtesy: Tulip Group of Companies)
Cultivation and isolation of bacteria from pathological
samples is many a times key to the identification of
the underlying infections. With ever-increasing strains
of resistant microorganisms, susceptibility testing to
antimicrobial agents complements selecting appropriate
drugs/drug regimens to treat infections.
Availability of microbiology testing and such procedures
being available in routine laboratories has been limited
due to the availability of dehydrated media, which can
be put to use only after substantial procedural and
preparatory requirements. INSTAPREP media are ready to
use/ready to pour and fill this long felt need using a unique
proprietary technology for routine microbiological testing.
MICROXPRESS INSTAPREP are reagents for laboratory
INSTAPREP is a ready to pour sterilized pouched media
for microbiological applications such as cultivation/
isolation/selective growth/susceptibility tests.
Nutrient agar is used as a general culture medium. It can
be used for maintaining microorganisms for prolonged
makes it suitable for cultivation of related fastidious
organisms. The poured medium is light straw colored,
slightly opalescent with a pH at 7.4 ± 0.2.
Microbiology and Bacteriology 829
MacConkey agar is the standard medium for the cultivation
of enterobacteria. It is a selective and differential medium.
It contains a bile salt to inhibit nonintestinal bacteria with
neutral red to distinguish the lactose fermenting coliforms
from the lactose non-fermenting Salmonella and Shigella
species. The poured medium is a distinct clear reddish
brown color with a pH at 7.4 ± 0.2.
Cysteine lactose electrolyte deficient (CLED) Agar
CLED agar with Andrade’s indicator is a medium of
choice, recommended for use in urinary bacteriology as it
promotes the growth of all urinary pathogens. Additionally
since, it is an electrolyte deficient medium swarming due
to Proteus species is prevented and direct colony count
is facilitated. For direct colony count, the medium is
inoculated by proper dilution of the sample. Additionally
CLED agar helps identify the organism directly from the
first isolate based on colony morphology and color within
24 hours. The poured medium is slightly opalescent
greenish/gray with a pH at 7.5 ± 0.2.
Sabouraud dextrose agar is the standard agar for the
cultivation and growth of fungi; particularly those
associated with skin infections. The poured medium is light
straw colored slightly opalescent with a pH at 5.6 ± 0.2.
Mueller Hinton agar is the standard agar recommended
for susceptibility tests using antibiotic sensitivity disks.
Mueller Hinton agar is recommended by NCCLS and WHO
Committee on standardization of susceptibility testing for
determining the susceptibility of microorganisms because
of its reproducibility. The poured medium is light amber
colored to slightly opalescent with a pH of 7.3 ± 0.1.
INSTAPREP ready to pour media are presterilized
media with standard proven formulations. The pouched
media only need to be kept in boiling (100oC) water for
10 minutes and they become ready to pour into sterile
plates. A result of Tulip’s long research the INSTAPREP
pouched media accord flexibility to the laboratories,
thereby avoiding laborious preparatory steps and wastage.
INSTAPREP media also help laboratories to set up cultures
on a random basis and not to be restricted to batching of
a. Store the pouches at room temperature (25 and 30oC).
b. Stability of the unopened pouch is as per the expiry
Water bath (250 mL beaker) at 100oC, vertical laminar air
flow/biosafety hood with Bunsen Burner, forceps/tongs,
sterile petridishes (disposable/glass), scissors, disinfectant
(70oC alcohol), absorbent sterile gauze, plastic/glass/wire
rod for hanging pouches in water bath.
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