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CULTURE MEDIA USED IN MICROBIOLOGYTuba BashirSupervisor Microbiology DepartmentTHE INDUS HOSPITAL, KARACHI
Story Behind Penicillin
The discovery of penicillin, one of the worlds first antibiotics, marks a true turning point in human history when doctors finally had a tool that could completely cure their patients of deadly infectious diseases.
Upon examining some colonies of Staphylococcus aureus, Dr. Fleming noted that a mold called Penicillium notatum had contaminated his Petri dishes. After carefully placing the dishes under his microscope, he was amazed to find that the mold prevented the normal growth of the staphylococci.
Definition, purpose/importanceHistory of culture mediaClassification of culture mediaGrowth pattern of bacteria Culture media are vital to microbiology , Without good media there is little chance that good results will emerge from the laboratory
Microbiological culture Method of cultivating microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions.
Bacteria have to be grown (cultured) for them to be identified and subsequent clinical diagnosis.By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study.Bacteria have to be cultured in order to obtain antigens from developing serological assay for vaccines.Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro.
Louis Pasteur used simple broths made up of urine or meat extracts.
Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media.History of culture medias
Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems. It existed as liquid at normal incubating temperatures (35-37oC) Digested by certain bacteria
AgarUsed for preparing solid mediumObtained from seaweeds.No nutritive valueNot affected by the growth of the bacteria.Melts at 98oC & sets at 42oC2% agar is employed in solid medium
Agar Fibers Agar Powder
Bacterial Growth Curve
Properties of Media:Support the growth of the bacteria.Should be nutritive (contains the required amount of nutrients).Suitable pH (neutral to slightly alkaline 7.3-7.4).Suitable temperature, and suitable atmosphere. (Bacteria grow at 370C)Note: media are sterilized by autoclaving at 1210C and 2 atmosphere for 15-20 minutes. With the autoclave, all bacteria, fungi, viruses, and spores are destroyed. Some media cant be sterilized by autoclaving because they contain eggs or carbohydrates .
Classification: Bacterial culture media can be classified in at least three ways 1.CONSISTANCY2.NUTRITIONAL COMPONENT 3.FUNCTIONAL USE
1) Classification based on consistency: A. Liquid media :
. . These are available for use in test-tubes, bottles or flasks. Liquid media are sometimes referred as broths
e.g nutrient broth BHIIn liquid medium, bacteria grow uniformly producing general turbidity
B.Solid media:An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganismsAgar is the most commonly used solidifying agent
C) Semi-solid agar: Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains
2) Classification based on nutritional component: Simple media:Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate Complex media:
specially prepared media for research purposes where the composition of every component is well known. Synthetic media:
3) Classification based on functional use or application: Basal mediaEnriched mediaSelective mediaEnrichment media Differential mediaTransport mediaAnaerobic mediaMedia for Biochemical Identification
3) Classification based on functional use or application:Basal media
Basal media are basically simple media that supports most non-fastidious bacteria
Examples of Basal media:Peptone water,nutrient broth and nutrient agar
3) Classification based on functional use or application:Enriched mediacontain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Examples of Enriched media: Blood agar Chocolate agar
Blood agar plate (BAP) Contains mammalian blood (usually sheep or horse), typically at a concentration of 510%. BAP are enriched, differential media used to isolate fastidious organisms and detect hemolytic activity
CHOCOLATE AGARChocolate agar - is a non-selective, enriched growth medium. containing red blood cells that have been lysed by slowly heating to 80 C. Chocolate agar is used for growing fastidious bacteria, such as Haemophilus influenzae
3) Classification based on functional use or application:Selective mediaAny agar media can be made selective by addition of certain inhibitory agents that dont affect the pathogen. To make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these
LOWEINSTON JENSON (L.J )IS A SOLID MEDIUM USED FOR MTB
Sabouraud Dextrose Agar (SDA)
Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
3) Classification based on functional use or application:Enrichment media liquid media that also serves to inhibit commensal in the clinical specimen.
Selenite F broth and alkaline peptone water are used to recover pathogens from fecal specimens.
3) Classification based on functional use or application:Differential mediaCertain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Example of differential mediaMacConkeys agar, CLED agar, XLD agar etc
MacConkey Agar culture medium designed to grow Gram-negative bacteria and differentiate them for lactose fermentation It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which also inhibits certain Gram-positive bacteria)
CYSTEINE LACTOSE ELECTROLYTE DIFFECIENT AGARC.L.E.D. Agar is a non selective solid medium for cultivation of pathogens from urine specimens. Lack of salts (electrolytes) inhibits swarming of Proteus sp.
With Andrades Indicator
BIGGYBismuth Sulfite Glucose Glycine Yeast Agar is a selective and differential medium used in the isolation and presumptive identification ofCandidasppExamples of Differential media
3) Classification based on functional use or application:Transport mediaClinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Example of Transport mediaCary Blair medium for campylobacter species
Alkaline peptone water medium for V. cholerae
Some important criteria of Transport mediaTransport media should fulfill the following criteria:temporary storage of specimens being transported to the laboratory for cultivation. maintain the viability of all organisms in the specimen without altering their concentration. contain only buffers and salt. lack of carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication. transport media used in the isolation of anaerobes must be free of molecular oxygen. Transport Swab features a 5 mL Amies agar gel column with oxygen-scavenging agents, for sampling of both aerobic and facultative anaerobic organisms.
3) Classification based on functional use or application:Anaerobic mediaAnaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation reduction potential and extra nutrients. Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K Example of Anaerobic mediaThioglycollate medium
Media for Biochemical Identification
it is a non-selective, non-differential mediumit contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibioticsit is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a true zone of inhibition.
Quality tests chemical and biological parameter checked to ensure end products meet product quality specification, packaging,labelling and storage are important