CULTURE MEDIA USEDCULTURE MEDIA USEDIN MICROBIOLOGYIN MICROBIOLOGYSHEIKH JONAID NIZAMITrainee TechnologistClinical LaboratoryAGA KHAN UNIVERSITYHOSPITAL KARACHI
Definition, purpose/importanceHistory of culture mediaClassification of culture mediaGrowth pattern of bacteria
Microbiological cultureMethod of cultivating microbialMethod of cultivating microbialorganisms by letting them reproduceorganisms by letting them reproducein predetermined culture media underin predetermined culture media undercontrolled laboratory conditions.controlled laboratory conditions.
Bacteria have to be grown (cultured) for themBacteria have to be grown (cultured) for themto be identified and subsequent clinicalto be identified and subsequent clinicaldiagnosis.diagnosis.By appropriate procedures they have to beBy appropriate procedures they have to begrown separately (isolated) on culture mediagrown separately (isolated) on culture mediaand obtained as pure for study.and obtained as pure for study.Bacteria have to be cultured in order to obtainBacteria have to be cultured in order to obtainantigens from developing serological assay forantigens from developing serological assay forvaccines.vaccines.Certain genetic studies and manipulations ofCertain genetic studies and manipulations ofthe cells also need that bacteria be cultured inthe cells also need that bacteria be cultured invitro.vitro.
Louis PasteurLouis Pasteur used simple broths made upused simple broths made upof urine or meat extracts.of urine or meat extracts. Robert KochRobert Kochrealized the importance of solid media andrealized the importance of solid media andusedused potato piecespotato pieces to grow bacteria.to grow bacteria. It wasIt wason the suggestion of Fannie Eilshemius,on the suggestion of Fannie Eilshemius,wife of Walther Hesse (who was anwife of Walther Hesse (who was anassistant to Robert Koch) that agar wasassistant to Robert Koch) that agar wasused to solidify culture media.used to solidify culture media.History of culture medias
Before the use of agar, attempts were made toBefore the use of agar, attempts were made touse gelatin as solidifying agent.use gelatin as solidifying agent. GelatinGelatin hadhadsome inherent problems….some inherent problems….It existed as liquid at normalIt existed as liquid at normalincubating temperatures (35-37incubating temperatures (35-37ooC)C)Digested by certain bacteriaDigested by certain bacteriaContinued….
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 bacteria.Not affected by the growth of the bacteria. Melts at 98Melts at 98ooC & sets at 42C & sets at 42ooCC 2% agar is employed in solid medium2% agar is employed in solid medium
During typical bacteria growth (growth cycle)bacteria cell divide by binary fission andtheir mass and number increase in anexponential manners. Bacterial growth inculture can be separated into at least fourdistinct phases.Bacterial Growth Curve
This is period of intense physiologic adjustmentinvolving the induction of new enzymes and thesynthesis and assembly of ribosome. In lag phaseand during this phase there occur1. increase in size of cells2. increase in metabolic rate3. adaptation to new environment andnecessary enzymes.1. Lag phase
The length of lag phase depend upona. Type of bacteria.b. Better the medium, shorter the lag phase.c. The phase of culture from which inoculation intakend. Size or volume of inoculum.e. Environmental factors like temperature.Continued….
2. Logarithmic (Exponential) phaseIn logarithmic phase the bacterial cellstart dividing and their number increase bygeometric progression with time. During thisperiods…a. bacteria have high rate of metabolismb. bacteria are more sensitive to antibioticsc. rate of penetration of the medium itdepends on the concentration of material inthe media
3. Stationary phaseIn stationary phase after some time a stagecomes when rate of multiplication and deathbecomes almost equal it may be due toa. depletion of nutrientb. accumulation of toxic products andsporulation may occur during this stage.
4. Decline or death phaseIn decline (death) phase, during this phasepopulation decreases due to death of cells thefactors responsible area. nutritional exhaustionb. toxic accumulationc. autolysis enzymes
TYPES OF CULTURE MEDIATYPES OF CULTURE MEDIAI.I. Based on their consistencyBased on their consistencya) solid mediuma) solid mediumb) liquid mediumb) liquid mediumc) semi solid mediumc) semi solid mediumII.II. Based on the constituents/ ingredientsBased on the constituents/ ingredientsa) simple mediuma) simple mediumb) complex mediumb) complex mediumc) synthetic or defined mediumc) synthetic or defined mediumd) Special mediad) Special media
Special mediaSpecial media Enriched mediaEnriched media Enrichment mediaEnrichment media Selective mediaSelective media Indicator mediaIndicator media Differential mediaDifferential media Transport mediaTransport mediaIII.III. Based on Oxygen requirementBased on Oxygen requirement- Aerobic media- Aerobic media- Anaerobic media- Anaerobic media
Solid mediaSolid media – contains 2% agar– contains 2% agar Colony morphology, pigmentation, hemolysis can beColony morphology, pigmentation, hemolysis can beappreciated.appreciated. Eg: Nutrient agar, Blood agarEg: Nutrient agar, Blood agarLiquid mediaLiquid media – no agar.– no agar. For inoculum preparation, Blood culture, for theFor inoculum preparation, Blood culture, for theisolation of pathogens from a mixture.isolation of pathogens from a mixture. Eg: Nutrient brothEg: Nutrient brothSemi solid mediumSemi solid medium – 0.5% agar.– 0.5% agar. Eg:Eg: SIMSIM
Simple media / basal mediaSimple media / basal media-- Eg: NB, NAEg: NB, NA- NB consists of peptone, yeast extract, NaCl,- NB consists of peptone, yeast 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 nutrientsProvide special nutrientsSynthetic or defined mediaSynthetic or defined media Media prepared from pure chemicalMedia prepared from pure chemicalsubstances and its exact composition issubstances and its exact composition isknownknown Eg: peptone water – 1% peptone + 0.5% NaClEg: peptone water – 1% peptone + 0.5% NaClin waterin water
Enriched mediaEnriched media Substances like blood, serum, egg areSubstances like blood, serum, egg areadded to the basal medium.added to the basal medium. Used to grow bacteria that are exacting inUsed to grow bacteria that are exacting intheir nutritional needs.their nutritional needs. Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar
Enrichment mediaEnrichment media Liquid media used to isolateLiquid media used to isolatepathogens from a mixed culture.pathogens from a mixed culture. Media is incorporated withMedia is incorporated withinhibitory substances toinhibitory substances tosuppress the unwantedsuppress the unwantedorganism.organism. Eg:Eg: Selenite F BrothSelenite F Broth – for the isolation– for the isolationof Salmonella, Shigellaof Salmonella, Shigella Alkaline Peptone WaterAlkaline Peptone Water – for Vibrio– for Vibriocholeraecholerae
Selective mediaSelective media The inhibitory substance is added to a solidThe inhibitory substance is added to a solidmedia.media.Eg:Eg: Mac Conkey’s mediumMac Conkey’s medium for gram negativefor gram negativebacteriabacteria TCBSTCBS – for V.cholerae– for V.cholerae LJ mediumLJ medium – M.tuberculosis– M.tuberculosis Wilson and Blair mediumWilson and Blair medium – S.typhi– S.typhi Potassium tellurite mediumPotassium tellurite medium – Diphtheria– Diphtheriabacillibacilli
Indicator mediaIndicator media These media contain an indicator whichThese media contain an indicator whichchanges its colour when a bacterium growschanges its colour when a bacterium growsin them.in them. Eg:Eg: Blood agarBlood agar Mac Conkey’s mediumMac Conkey’s medium Christensen’s urease mediumChristensen’s urease medium
Differential mediaDifferential media A media which has substances incorporatedA media which has substances incorporatedin it enabling it to distinguish betweenin it enabling it to distinguish betweenbacteria.bacteria. Eg:Eg: Mac Conkey’s mediumMac Conkey’s medium Distinguish between lactose fermenters & nonDistinguish between lactose fermenters & nonlactose fermenters.lactose fermenters.
Transport mediaTransport media Media used for transporting theMedia used for transporting thesamples.samples. Delicate organisms may notDelicate organisms may notsurvive the time taken forsurvive the time taken fortransporting the specimentransporting the specimenwithout a transport media.without a transport media. Eg:Eg: Stuart’s mediumStuart’s medium – non nutrient soft– non nutrient softagar gel containing a reducingagar gel containing a reducingagentagent Buffered glycerol salineBuffered glycerol saline – enteric– entericbacillibacilli
Anaerobic mediaAnaerobic media These media are used to grow anaerobic organisms.These media are used to grow anaerobic organisms. Eg:Eg: Robertson’s cooked meat medium, ThioglycolateRobertson’s cooked meat medium, Thioglycolatemedium.medium.