CONTENTS

1.INTRODUCTION

2.HISTORY

3.CULTIVATION

4.EQUIPMENT

5.MEDIA CLASSIFICATION AND

FUNCTION

6.FREQUENTLY USED MEDIA

7.TYPES OF CULTURE MEDIA

8.CULTURE METHODS

9.CONCLUSION

10.REFERENCES

INTRODUCTION:-

Direct laboratory methods, such as microscopy provide

preliminary information about the bacteria involved in an

infection, but bacterial growth is usually required for definitive

identification and characterization,hence the culture method

is needed.

HISTORY

Hippocrates is credited with being the first person to believe

that diseases were caused naturally, not because of

superstition and gods.

Antonie Van Leeuwenhoek (1632–1723) was one of the first

person to observe microorganisms using a microscope of his

own design and named them as “little beasties”

Robert Hooke was the first person who made key observations

on living things with the use a microscope.

• Lazzaro Spallanzani (1729–1799) found that boiling broth

would sterilise it and kill any microorganisms in it.

• Louis Pasteur and the germ theory of disease, which states

that microorganisms are the causes of infectious disease.

• In 1876, Robert Koch (1843–1910) established that microbes

can cause disease.

CULTIVATION:

Cultivation is the process of growing

microorganisms in culture by taking bacteria from

the infection site (i.e., the in vivo environment) by

some means of specimen collection and growing

them in the artificial environment of the laboratory

(i.e., the in vitro environment).

PRINCIPLES OF BACTERIAL CULTIVATION:

● To grow and isolate all bacteria present in a clinical

specimen

● To determine which of the bacteria that grow are most

likely causing infection and which are likely contaminants

or colonizers

● To obtain sufficient growth of clinically relevant bacteria

to allow identification and characterization

EQUIPMENT :

1.Petri dish :

2.Bunsen Burner :

3.Inoculating loop :

Need for Culture media:

Used to grow bacteria

Can be used to enrich the numbers of bacteria

Select for certain bacteria and suppress others

Differentiate among different kinds of bacteria

WATER.CARBON SOURCE.NITROGEN SOURCE.INORGANIC SALTS

Phosphates. Sulfates.Sodium. Potassium.Magnesium. Iron.Manganese. Calcium.

BACTERIAL VITAMINS

Thiamine riboflavinnicotinic acid pyridoxinefolic acid vitamine B12

BASIC NUTRITIONAL REQUIREMENTS

GASEOUS REQUIREMENTS

OXYGEN

Obligate aerobesFacultative anaerobes Microaerophillic.Obligate anaerobes.

.

CARBONDIOXIDE:

Capnophillic

TEMPERATURE

• Psychrophiles: with optimum growth T

around 20 C

• Mesopihles: between 15 and 45 with

optimum around 37 C

• Thermophiles: between 30 and 75 with

optimum around 55 C

• Hyperthermophiles: T grater than 100C

pH

• Most bacteria grow between pH 6.5 and

7.5

• Molds and yeasts grow between pH

5 and 6

COLONIES - MAKE A OBSERVATION

• Shape

• Size

• Elevation

• Edge

• Surface

• Opacity

• Consistency

Types of culture media

1.Solid

2.liquid

Liquid medium:

• bacteria in small numbers grow

only in liquid media e.g. blood

culture.

• used for biochemical test

• Earliest liquid medium: urine or

meat broth - Louis Pasteur.

• Isolation and identification of

bacteria in pure culture is not

possible.

Solid medium:

• Distinct colony morphology

• Characteristics → easy to

identify

• Colony – macroscopically

visible collection of millions

of bacteria originating from

a single bacterial cell

Earliest solid medium:

Cooked cut potato by Robert Koch

Gelatin - not satisfactory

- liquefy at 24oC

Agar

Frau Hesse

Universally used for preparing solid medium

Obtained from seaweed: Gelidium

No nutritive value

Long chain polysaccharides.

- Also contains varying amounts of inorganic salts and

small quantities of a protein – like substance

Not affected by the growth of the bacteria.

Melts at 98°C & sets at 42°C

2% agar is employed in solid medium

FREQUENTLY USED MEDIA:

Brain-Heart Infusion.

Sheep Blood Agar.

Chocolate Agar.

Columbia CNA with Blood.

MacConkey Agar.

Thioglycollate Broth.

Brain-Heart Infusion Nutritionally rich medium

Allows the growth of aerobic organisms

Key ingredients:

Infusion from several animal tissue sources

Added peptone (protein)

Phosphate buffer

Dextrose

uses :1. a major component of the media developed for culturing a patient’s blood for bacteria

2. to determine bacterial susceptibility to antimicrobial agents

An enrichment media for

certain fastidious bacteria like N.gonorrhoeae, haemophilous spp

These bacteria utilize

haemoglobin,

hemin(x factor), coenzyme

Nicotinamide adenine

dinucleotide,

(v factor) released from lysis of

RBC

Red blood cell lysis gives the

medium a chocolate-brown

colour

from which this agar gets its

name

Chocolate Agar

MacConkey Agar Frequently used primary selective

and differential medium

Ingredients:

crystal violet: supresses +ve & fungi

neutral red: pH indicator provide this medium with a differential capacity

MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red to pink.

Lactose fermenters – Pinkcolonies

Non lactose fermenters –colourless colonies

eg: Shigella spp.

Supportive medium for the

isolation and differentiation of

gram +ve cocci from clinical and

non-clinical specimens which

contain mixed flora

Three peptone sources

5% defibrinated sheep blood

Colistin

Nalidixic acids

can also be used to help

differentiate bacterial colonies

based on the hemolytic reactions

they produce

To suppress gram –

ve

Columbia CNA with Blood

Sheep Blood Agar. supports all but the most fastidious clinically significant bacteria

It has specific colony morphology which is easier to identify bacteria

Ingredients: tryptons

soyabean protein digest

NaCl

Agar

5% sheep blood

extracellular enzymes that lyse

red blood cells in the agar (hemolysis).

Alpha hemolysis

partial lysis --greenish discoloration around the colony.

Beta hemolysis

complete clearing of the red blood cells around the bacterial colony

gamma or nonhemolysis

no halo is produced around the colony

Thioglycollate Broth Most frequently used enrichment

medium

Ingredients:

1.nutrient factors, including casein,

yeast and beef extracts, and vitamins.

2. oxidation-reduction indicator

(resazurin), dextrose, phylloquinone

and hemin .

3. 0.075% agar to prevent water

currents to take O2 in to deeper layers.

Agar & thioglycollate (reducing

agent) allow anaerobic bacteria to

grow deeper in the tube.

Gram-positive cocci grow as“puff

balls.”

Strictly aerobic organisms such as

P.aeruginosa, grow toward the top of

the broth

Strictly anaerobic organisms grow in

the bottom of the broth

Types of culture media

I. Based on their consistency

a) Solid medium

b) Liquid medium

c) Semi solid medium

II. Based on the constituents/

ingredients

a) Simple medium

b) Complex medium

c) Synthetic or defined medium

d) Special media

Special media

Enriched media

Enrichment media

Selective media

Indicator media

Differential media

Sugar media

Transport media

Media for biochemical reactions

III.Based on Oxygen

requirement

- Aerobic media

- Anaerobic media

Simple media / basal media:

Most common in routine diagnostic laboratories

Eg: Nutrient Broth, Nutrient Agar

NB consists of peptone, meat extract, NaCl, water

NB + 0.5% Glucose = Glucose Broth

NB + 2% agar = Nutrient agar

Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium

Complex media

Media other than basal media.

They have added complex ingredients such as yeast extract or

casein hydrolysate, which consist of a mixture of many

chemical species in unknown proportions

Provide special nutrients

Synthetic or defined media

Media prepared from pure chemical substances

exact composition is known

Used for special studies, eg. metabolic requirements

Eg: peptone water- (1% peptone + 0.5% NaCl in water)

Enriched media

Substances like blood, serum, egg are added to the basal

medium.

Used to grow bacteria that are exacting in their nutritional

needs.

Eg: Blood agar, Chocolate agar

Chocolate

agar

Blood agar

Enrichment media

Liquid media used to isolate pathogens from a mixed culture.

Media is incorporated with inhibitory substances to suppress

the unwanted organism → increase in numbers of desired bacteria

Eg:

Selenite F Broth –for the isolation of salmonella,Shigella

Tetrathionate Broth ( inhibit coliforms)

Alkaline Peptone Water – for Vibrio cholerae

Selective media

The inhibitory substance is added to a solid media

Increase in number of colonies of desired bacterium

Eg:

Desoxycholate citrate medium for dysentery bacilli

Mac Conkey’s medium for gram negative bacteria

Thiosulfate citrate bile salts sucrose (TCBS) agar– for V.

cholerae

LJ medium – M. tuberculosis

Thiosulfate citrate

bile salts sucrose (TCBS) agarMac Conkey’s medium

Deoxycholate citrate agar LJ media

Indicator media

contain an indicator which changes its colour when a bacterium

grows in them

Eg:

Wilson-Blair medium – S. typhi forms black colonies

McLeod’s medium (Potassium tellurite)– Diphtheria bacilli

Wilson-Blair Medium McLeod’s medium

• Differential media

some factor that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate.

• Eg:

MacConkey agar:

A. Deep purple

B. Light pink or colourless

Sugar media

Media containing any fermentable substance

Eg: glucose, arabinose, lactose, starch etc.

Media consists:

1% of the sugar in peptone water + Indicator

Contain a small tube (Durham’s tube) for the detection of gas by

the bacteria

Transport media

Media used for transporting the samples.

Delicate organisms may not survive the time taken for

transporting the specimen without a transport media.

Eg:

Stuart’s medium – non nutrient soft agar gel containing a

reducing agent & charcoal

used for Gonnococci

Buffered glycerol saline – enteric bacilli

Anaerobic media

These media are used to grow anaerobic organisms.

Eg: Robertson’s cooked meat medium, Thioglycolate

medium.

CULTURE METHODS

Culture methods employed depend on the purpose for

which they are intended.

Purposes:

Isolation

Properties of bacteria

To create antigens for

laboratory use

To test for Antibiotic sensitivity

Estimate viable counts

Maintain stock cultures

Culture methods include:

Streak culture

Lawn culture

Stroke culture

Stab culture

Pour plate method

Liquid culture

Anaerobic culture methods

STREAK CULTURE

Used for the isolation of bacteria in pure

culture from clinical specimens.

Platinum wire is used.

One loopful of the specimen is transferred

onto the surface of a well dried plate.

Spread over a small area at the periphery.

The inoculum is then distributed thinly over

the plate by streaking it with a loop in a

series of parallel lines in different

segments of the plate.

On incubation, separated colonies are

obtained over the last series of streaks.

LAWN CULTURE

Provides a uniform surface growth

of the bacterium.

Uses

For bacteriophage typing.

Antibiotic sensitivity testing.

In the preparation of bacterial antigens and vaccines.

Lawn cultures are prepared by flooding the surface of the plate

with a liquid suspension of the bacterium.

Antibiotic sensitivity testing

STROKE CULTURE

Stroke culture is made in tubes containing

agar slope.

Uses

Provide a pure growth of bacterium for slide

agglutination and other diagnostic tests.

STAB CULTURE

Prepared by puncturing a suitable medium – gelatin or glucose

agar with a long, straight, charged wire.

Uses

Demonstration of gelatin liquefaction.

Oxygen requirements of the bacterium under study.

Maintenance of stock cultures.

POUR PLATE CULTURE

Agar medium is melted (15 ml) and cooled to 45oC.

1 ml of the inoculum is added to the molten agar.

Mix well and pour to a sterile petri dish.

Allow it to set.

Incubate at 37oC, colonies will be distributed throughout the

depth of the medium.

Uses

Gives an estimate of the viable bacterial count in a

suspension.

For the quantitative urine cultures.

LIQUID CULTURES

Liquid cultures are inoculated by touching with a charged

loop or by adding the inoculum with pipettes or syringes.

Uses

Blood culture

Sterility tests

Continuous culture methods

Disadvantage

It does not provide a pure culture

from mixed inocula

ANAEROBIC CULTURE METHODS

Anaerobic bacteria differ in their requirement and sensitivity to

oxygen.

Cl. tetani is a strict anaerobe - grows at an oxygen tension < 2 mm

Hg.

Methods:

Production of vacuum

Displacement of oxygen with other gases

Chemical method

Biological method

Reduction of medium

Displacement of oxygen with other gases

Displacement of oxygen with hydrogen, nitrogen, helium or

CO2.

Eg: Candle jar

Inoculated plates are placed inside a large airtight container

And a lighted candle kept in it before the lid is sealed

The burning candle is expected to use up all the oxygen inside

Before it is extinguished ,but some oxygen is always left behind.

The candle jar provides a concentration of carbondioxide which

stimulates the growth of most bacteria.

Chemical method

Alkaline pyrogallol absorbs oxygen.pyrogallic acid added to a solution

of sodium hydroxide in a large test tubes placed inside an airtight jar povides

anaerobic environment but small amount of carbonmonoxide, which is formed

during reaction,may be inhibitory to some bacteria

Instead of alkaline pyrogallol,anaerobic environment has been produced

within jars with a mixture of Chromium and Sulphuric acid

McIntosh – Fildes’ anaerobic jar

Consists of a metal jar or glass jar with a metal lid

which can be clamped air tight.

The lid has 2 tubes – gas inlet and gas outlet

The lid has two terminals – connected to electrical supply.

Under the lid – small grooved porcelain spool, wrapped

with a layer of palladinised asbestos.

Working:

Inoculated plates are placed inside the jar and the lid

clamped air tight.

The outlet tube is connected to a vacuum pump and the air

inside is evacuated.

The outlet tap is then closed and the inlet tube is connected to a

hydrogen supply.

After the jar is filled with hydrogen, the electric terminals are

connected to a current supply, so that the palladinised asbestos

is heated.

Act as a catalyst for the combination of hydrogen with residual

oxygen.

Gaspak

Commercially available disposable envelope.

Contains chemicals which generate H2 and CO2 on addition of

water.

Cold catalyst – permits combination

of Hydrogen & Oxygen

Indicator is used – reduced methylene

blue.

Colourless – anaerobically

Blue colour – on exposure to oxygen

Biological method

Absorption of oxygen by incubation with aerobic bacteria,

germinating seeds or chopped vegetables.

Reduction of oxygen

By using reducing agents – 1% glucose, 0.1% Thioglycolate

CONCLUSION

Even though there are several methods for bacterial

identification, culture method of cultivating microorganisms is

considered the gold standard procedure because we can

isolate pure culture of bacteria in viable condition, which is a

major drawback with PCR.

1.BEILY & SCOTT diagnostic microbiology 12th edition.

2.R.ANANTHANARAYAN AND C.K.J PANIKER Text book of microbiology 7th

edition

3.mim’s MEDIAL MICROBIOLOGY 4th edition.

REFERENCES