avoided. Storage and Stability a. Store the pouches at room temperature (25 and 30oC). b. Stability of the unopened pouch is as per the expiry

 

Serology

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.

Preparation of Culture Media

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:

Peptone Water

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

cholerae.

Peptone 10 g

Sodium chloride 5 g

Distilled water 1000 mL

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.

Nutrient Broth

Peptone 10 g

Sodium chloride 5 g

Meat extract 10 g

Distilled water 1000 mL.

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.

Nutrient Agar

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

cooled.

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

20 minutes.

Blood Agar

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.

Chocolate Agar

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

needed.

Sugar Media

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—

pink color.

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.

Hiss’s Serum Water Sugars

This is used for biochemical reaction of Neisseriae.

Corynebacterium and other organisms requiring serum

for growth.

Ox serum 1 part

Distilled water 3 part

Adjust reaction to pH 7.5 and Andrades indicator 1% and

sugar 1%. Sterilize as for peptone water/sugar media.

Loeffler’s Serum Slopes

(Used for cultivating diphtheria bacilli)

Ox serum 3 parts

Glucose broth 1% 1 part

Blood is collected from ox under sterile conditions and

serum separated aseptically. Inspissate the medium at

75oC for 1 hour.

Media for Identification of Fungi

Sabouraud’s Glucose Agar

Glucose 40 grams

Peptone 10 grams

Agar 20 grams

Water to 1000 mL

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

INSTAPREP

(Courtesy: Tulip Group of Companies)

Summary

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.

Reagent

MICROXPRESS INSTAPREP are reagents for laboratory

use only.

INSTAPREP is a ready to pour sterilized pouched media

for microbiological applications such as cultivation/

isolation/selective growth/susceptibility tests.

Nutrient Agar

Nutrient agar is used as a general culture medium. It can

be used for maintaining microorganisms for prolonged

survival of cultures. It can be used for cultivation of nonfastidious organisms. Addition of sheep blood/or serum

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

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

with Andrade’s Indicator

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

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

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.

Principle

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

cultures. As compared to prepoured plates and dehydrated media, variability, contamination and wastage is also

avoided.

Storage and Stability

a. Store the pouches at room temperature (25 and 30oC).

b. Stability of the unopened pouch is as per the expiry

date mentioned on the label.

Additional Material Required

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.

Procedures

1. Retrieve the required number of pouches from the

carton.