Bacteriological Culture Media

Objectives To be familiar with different types of culture

media used in a microbiology laboratory. To be familiar with the pure technique for

pouring sterilized media into Petri dishes and tubes .

CultureA culture is the microorganisms that grow in

a culture medium. To culture means to grow microorganisms in

culture media.Culture media are nutrient-rich

substances that are used to grow bacteria in a lab setting .

History:

Louis Pasteur used simple broths made up of urine or meat extracts.

Robert Koch used potato pieces to grow bacteria as earliest solid medium

Gelatin was attempted to be used as the solid media , but was not satisfactory : liquefied at 35-37oC digested by certain bacteria.

Frau Hesse used agar for preparing solid medium.Agar is obtained from seaweeds.No nutritive valueNot affected by the growth of the bacteria.Melts at 95oC & solidifies at 42oC

Culture mediaSuitable quality culture media is required for

the successful isolation of bacteriaCulture media can be used to:

Enrich the numbers of bacteria.Select for certain bacteria and suppress others.Differentiate among different kinds of bacteria.

Classification:Culture media can be classified based on Consistency: Liquid – broth - e.g. Nutrient BrothSolid - agar ( 1 - 3%) - e.g. Nutrient agarSemisolid - agar (0.2 -0.5%) –e.g. Motility

medium

ClassificationClassification based on functional use or

application:

Ordinary /simple /basal media Support most non-fastidious bacteria e.g : NB, NA

Enriched media

Media that have been supplemented with highly nutritious materials for the purpose of cultivating fastidious organisms.

Substances like blood, serum, egg yolk are added to the basal medium(broth or solid )

e.g: Blood agar, Chocolate agar

Blood Agar media

Is an enriched complex medium with non-selective enrichment (Blood), as it allows a wide variety of organisms to grow including the fastidious ones

5% sheep’s blood + a complex medium containing peptones

Blood agar Chocolate agar

Haemophilus influenzae on Chocolate Agar Plate

Types of Hemolysis

Selective Enrichment media

Media is incorporated with inhibitory substances to suppress the unwanted organism.

Various approaches to make a medium selective include: addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these.

e.g:Selenite F Broth – for Salmonella, Shigella Alkaline Peptone Water – for Vibrio cholerae

Selective EnrichmentSelenite Broth medium A liquid media containing chemical materials

which inhibit some normal flora and allow pathogens (Salmonella and Shigella) which may be present in very small numbers in the stool specimen to grow .

Differential media Distinguish one microorganism type from another

growing on the same media. This type of media uses the biochemical

characteristics of a microorganism growing in the presence of specific nutrients or indicators .

Eg:Mac Conkey’s - Selective and Differential for G –ve

bacteriaMannitol Salt Agar - Selective and Differential for

S .aureus.

Mac Conkey agarSelective Media –

for gram negative enteric rods .Contains crystal violet and bile salts which

inhibit gram positive bacteria Differential Media

Contains lactose as a substrate and neutral red as an indicator Lactose fermentors – bacteria appear pink Non lactose fermentors - no change of colour

MacConkey’s agar with lactose fermenters (left) and non-lactose fermenters (right)

Mannitol Salt AgarSelective Media

For gram positive bacterium StaphylococciContains 7.5% NaCl as the inhibitor agent.

• Differential Media Contains Mannitol as the substrate and phenol red as

the pH indicatorMannitol fermentor – yellow colonies with yellow

zones,Mannitol non –fermentor- no color change

MSA

Staphylococcus aureus  on MSA

Staphylococcus epidermidis on MSA

Transport media Used to sustain the viability of organisms

while a clinical specimen is transported to the lab

Maintain the pathogen to commensal ratio e.g. Stuart medium –

non nutritional semi-solid media

SterilizationTransmissible agents (such as spores,

bacteria and viruses) can be eliminated through sterilization. 

The efficiency of the sterilization process depends on two major factors. the thermal death time – the thermal death point or temperature at

which all microbes in a sample are killed-

AutoclavingThe preferred principle for sterilization is

through heat under high pressure with steam. The autoclave being the most widely used method of achieving it.

For most types of media and other materials, steam- under-pressure sterilization is used .

Such material is autoclaved at 121°C for 15-20 minutes using steam under 15 pounds pressure.

Autoclave Design and ControlTo be effective against spore forming bacteria

and viruses, autoclaves need to:Have steam in direct contact with the

material being sterilized .Create vacuum in order to displace all the air

initially present in the autoclave and replacing it with steam.

Implement a well designed control scheme for steam evacuation and cooling so that the load does not perish.

AutoclaveProcess performance can be confirmed by

Monitoring colour changes on indicator tape taped onto materials to be autoclaved

Using Biological indicators such as the Attests e.g. Bacillus sterothermophilus spores .