CULTURE MEDIA USED CULTURE MEDIA USED IN MICROBIOLOGYIN MICROBIOLOGY

SHEIKH JONAID NIZAMITrainee Technologist Clinical Laboratory

AGA KHAN UNIVERSITY HOSPITAL KARACHI

Definition, purpose/importance

History of culture media

Classification of culture media

Growth pattern of bacteria

Microbiological culture Method of cultivating microbial Method of cultivating microbial

organisms by letting them organisms by letting them reproduce in predetermined reproduce in predetermined culture media under controlled culture media under controlled laboratory conditions.laboratory conditions.

Bacteria have to be grown (cultured) for Bacteria have to be grown (cultured) for them to be identified and subsequent them to be identified and subsequent clinical diagnosis.clinical diagnosis.By appropriate procedures they have to By appropriate procedures they have to be grown separately (isolated) on be grown separately (isolated) on culture media and obtained as pure for culture media and obtained as pure for study.study.Bacteria have to be cultured in order to Bacteria have to be cultured in order to obtain antigens from developing obtain antigens from developing serological assay for vaccines.serological assay for vaccines.Certain genetic studies and Certain genetic studies and manipulations of the cells also need that manipulations of the cells also need that bacteria be cultured in vitro.bacteria be cultured in vitro.

Louis PasteurLouis Pasteur used simple broths used simple broths made up of urine or meat extracts. made up of urine or meat extracts. Robert Koch Robert Koch realized the importance realized the importance of solid media and used of solid media and used potato potato piecespieces to grow bacteria. to grow bacteria. It was on the It was on the suggestion of Fannie Eilshemius, wife of suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Walther Hesse (who was an assistant to Robert Koch) that agar was used to Robert Koch) that agar was used to solidify culture media.solidify culture media.

History of culture medias

Before the use of agar, attempts were Before the use of agar, attempts were made to use gelatin as solidifying made to use gelatin as solidifying agent. agent. GelatinGelatin had some inherent had some inherent problems….problems…. It existed as liquid at normal It existed as liquid at normal incubating temperatures (35-37incubating temperatures (35-37ooC) C) Digested by certain bacteriaDigested by certain bacteria

Continued….

AgarAgar Used for preparing solid mediumUsed for preparing solid medium Obtained from seaweeds.Obtained from seaweeds. No nutritive valueNo nutritive value Not affected by the growth of the Not affected by the growth of the

bacteria.bacteria. Melts at 98Melts at 98ooC & sets at 42C & sets at 42ooCC 2% agar is employed in solid medium 2% agar is employed in solid medium

During typical bacteria growth (growth cycle) bacteria cell divide by binary fission and their mass and number increase in an exponential manners. Bacterial growth in culture can be separated into at least four distinct phases.

Bacterial Growth Curve

Bacterial Growth Curve

This is period of intense physiologic adjustment involving the induction of new enzymes and the synthesis and assembly of ribosome. In lag phase and during this phase there occur 1. increase in size of cells 2. increase in metabolic rate 3. adaptation to new environment and necessary enzymes.

1. Lag phase

The length of lag phase depend upon

a. Type of bacteria.

b. Better the medium, shorter the lag phase.

c. The phase of culture from which inoculation in taken

d. Size or volume of inoculum.

e. Environmental factors like temperature.

Continued….

2. Logarithmic (Exponential) phase

In logarithmic phase the bacterial cell start dividing and their number increase by geometric progression with time. During this periods…

a. bacteria have high rate of metabolism

b. bacteria are more sensitive to antibiotics

c. rate of penetration of the medium it depends on the concentration of material in the media

3. Stationary phase

In stationary phase after some time a stage comes when rate of multiplication and death becomes almost equal it may be due to

a. depletion of nutrient

b. accumulation of toxic products and sporulation may occur during this stage.

4. Decline or death phase

In decline (death) phase, during this phasepopulation decreases due to death of cells the factors responsible are

a. nutritional exhaustion

b. toxic accumulation

c. autolysis enzymes

TYPES OF CULTURE MEDIATYPES OF CULTURE MEDIA

I.I. Based on their consistency Based on their consistency

a) solid mediuma) solid medium

b) liquid mediumb) liquid medium

c) semi solid mediumc) semi solid medium

II.II. Based on the constituents/ Based on the constituents/ ingredientsingredients

a) simple mediuma) simple medium

b) complex mediumb) complex medium

c) synthetic or defined mediumc) synthetic or defined medium

d) Special mediad) Special media

Special mediaSpecial media Enriched mediaEnriched media Enrichment mediaEnrichment media Selective mediaSelective media Indicator mediaIndicator media Differential mediaDifferential media Transport mediaTransport media

III.III. Based on Oxygen requirementBased on Oxygen requirement

- Aerobic media- Aerobic media

- Anaerobic media- Anaerobic media

Solid media Solid media – contains 2% agar– contains 2% agar Colony morphology, pigmentation, hemolysis Colony morphology, pigmentation, hemolysis

can be appreciated.can be appreciated. Eg: Nutrient agar, Blood agarEg: Nutrient agar, Blood agar

Liquid media Liquid media – no agar. – no agar. For inoculum preparation, Blood culture, for For inoculum preparation, Blood culture, for

the isolation of pathogens from a mixture.the isolation of pathogens from a mixture. Eg: Nutrient brothEg: Nutrient broth

Semi solid medium Semi solid medium – 0.5% agar. – 0.5% agar. Eg: Eg: SIMSIM

Simple media / basal media Simple media / basal media

- - Eg: NB, NAEg: NB, NA

- NB consists of peptone, yeast - NB consists of peptone, yeast extract, NaCl, extract, NaCl,

- - NB + 2% agar = Nutrient agarNB + 2% agar = Nutrient agar

Complex mediaComplex media Media other than basal media.Media other than basal media. They have added ingredients.They have added ingredients. Provide special nutrients Provide special nutrients

Synthetic or defined mediaSynthetic or defined media Media prepared from pure chemical Media prepared from pure chemical

substances and its exact composition substances and its exact composition is knownis known

Eg: peptone water – 1% peptone + Eg: peptone water – 1% peptone + 0.5% NaCl in water0.5% NaCl in water

Enriched mediaEnriched media

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

Used to grow bacteria that are Used to grow bacteria that are exacting in their nutritional needs.exacting in their nutritional needs.

Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar

Blood agar Chocolate agar

Enrichment media Enrichment media Liquid media used to isolate Liquid media used to isolate

pathogens from a mixed pathogens from a mixed culture.culture.

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

Eg: Eg: Selenite F Broth Selenite F Broth – for the – for the

isolation of Salmonella, isolation of Salmonella, Shigella Shigella

Alkaline Peptone Water Alkaline Peptone Water – for – for Vibrio choleraeVibrio cholerae

Selective mediaSelective media The inhibitory substance is added to a The inhibitory substance is added to a

solid media.solid media.

Eg:Eg: Mac Conkey’s mediumMac Conkey’s medium for gram for gram

negative bacterianegative bacteria TCBSTCBS – for V.cholerae– for V.cholerae LJ medium LJ medium – M.tuberculosis– M.tuberculosis Wilson and Blair mediumWilson and Blair medium – S.typhi – S.typhi Potassium tellurite medium Potassium tellurite medium – –

Diphtheria bacilliDiphtheria bacilli

TCBSMac Conkey’s medium

Potassium Tellurite media LJ media

Indicator mediaIndicator media These media contain an indicator These media contain an indicator

which changes its colour when a which changes its colour when a bacterium grows in them.bacterium grows in them.

Eg: Eg: Blood agarBlood agar Mac Conkey’s mediumMac Conkey’s medium Christensen’s urease mediumChristensen’s urease medium

Urease mediumUrease medium

Differential mediaDifferential media A media which has substances A media which has substances

incorporated in it enabling it to incorporated in it enabling it to distinguish between bacteria.distinguish between bacteria.

Eg: Eg: Mac Conkey’s mediumMac Conkey’s medium Distinguish between lactose Distinguish between lactose

fermenters & non lactose fermenters.fermenters & non lactose fermenters.

Lactose fermenters – Lactose fermenters – PinkPink colonies colonies Non lactose fermenters – colourless Non lactose fermenters – colourless

coloniescolonies

Transport mediaTransport media Media used for transporting Media used for transporting

the samples.the samples. Delicate organisms may not Delicate organisms may not

survive the time taken for survive the time taken for transporting the specimen transporting the specimen without a transport media.without a transport media.

Eg: Eg: Stuart’s medium Stuart’s medium – non – non

nutrient soft agar gel nutrient soft agar gel containing a reducing agentcontaining a reducing agent

Buffered glycerol saline Buffered glycerol saline – – enteric bacilli enteric bacilli

Anaerobic mediaAnaerobic media These media are used to grow anaerobic These media are used to grow anaerobic

organisms.organisms. Eg: Eg: Robertson’s cooked meat medium, Robertson’s cooked meat medium,

Thioglycolate medium.Thioglycolate medium.