CULTURE MEDIA & CULTURE METHODS

[Audio] CULTURE MEDIA USED IN MICROBIOLOGY Tuba Bashir Medical Researcher/ Writer.

[Audio] Story Behind Penicillin (This story tells how culture media examination is important) The discovery of penicillin, one of the world's 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..

. . Penicillium Area Of inhibition Of bacterial growth Normal bacterial.

[Audio] Definition, purpose/ importance History of culture media Classification of culture media Growth pattern of bacteria Culture media are vital to microbiology , Without good media there is little chance that good results will emerge from the laboratory.

[Audio] Microbiological culture Method of cultivating microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions..

[Audio]  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..

[Audio] 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.

[Audio] 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.

[Audio] Agar Used for preparing solid medium Obtained from seaweeds. No nutritive value Not affected by the growth of the bacteria.  Melts at 98oC & sets at 42oC  2% agar is employed in solid medium  Agar Fibers Agar Powder.

. . The OXO 1 D Manual THIRD EDITION. WATEF?.: COUNT TRIPLE suGZi.

[Audio] Bacterial Growth Curve. . . STATIONARY PHASE LOG PHASE DEATH PHASE LAG PHASE TIME.

[Audio] 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 can't be sterilized by autoclaving because they contain eggs or carbohydrates ..

[Audio] Classification: Bacterial culture media can be classified in at least three ways 1. CONSISTANCY 2.NUTRITIONAL COMPONENT 3.FUNCTIONAL USE.

[Audio] 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 BHI In liquid medium, bacteria grow uniformly producing general turbidity.

[Audio] B. Solid media: An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganisms Agar is the most commonly used solidifying agent.

[Audio] C) Semi-solid agar: Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains e.g: SIM.

[Audio] 2) Classification based on nutritional component: Simple media: Simple media such as peptone water, nutrient agar can support most nonfastidious 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:.

[Audio] 3) Classification based on functional use or application: Basal media Enriched media Selective media Enrichment media Differential media Transport media Anaerobic media Media for Biochemical Identification.

[Audio] 3) Classification based on functional use or application: • Basal media Basal media are basically simple media that supports most nonfastidious bacteria Examples of Basal media: Peptone water, nutrient broth and nutrient agar.

[Audio] 3) Classification based on functional use or application: Enriched media contain 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.

[Audio] BLOOD AGAR • Blood agar plate ( BAP) Contains mammalian blood (usually sheep or horse), typically at a concentration of 5– 10%. BAP are enriched, differential media used to isolate fastidious organisms and detect hemolytic activity.

[Audio] CHOCOLATE AGAR • Chocolate 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.

. . Staphylococcus aureus Haemophilus. inflåenza€..

[Audio] 3) Classification based on functional use or application: Selective media Any agar media can be made selective by addition of certain inhibitory agents that don't affect the pathogen. To make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these.

[Audio] • LOWEINSTON JENSON ( L.J )IS A SOLID MEDIUM USED FOR MTB • Sabouraud Dextrose Agar ( SDA) • Thiosulfate-citrate bile salts- sucrose agar ( TCBS).

[Audio] 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..

[Audio] 3) Classification based on functional use or application: Differential media Certain 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 media MacConkey's agar, CLED agar, XLD agar etc.

[Audio] 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).

[Audio] Lactose fermentors on MacConkeys agar Klebsiella pneumoniae on MacConkeys agar pink color colonies due to its lactose fermentation, and mucoid appearance due to its capsulated feature) E.coli on MacConkeys agar with dry pink color colonies due to its lactose fermentation Plain MacConkeys agar plate.

[Audio] Non lactose fermentors on MacConkeys agar Proteus mirabilis Pseudomonas aeroginosa with green color pigment Guess and comment (its pigment is a characteristic feature).

[Audio] CYSTEINE LACTOSE ELECTROLYTE DIFFECIENT AGAR • C.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.

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[Audio] BIGGY Bismuth Sulfite Glucose Glycine Yeast Agar is a selective and differential medium used in the isolation and presumptive identification of Candida spp Examples of Differential media.

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[Audio] 3) Classification based on functional use or application: Transport media Clinical 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 media Cary Blair medium for campylobacter species Alkaline peptone water medium for V. cholerae.

[Audio] Some important criteria of Transport media Transport 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 oxygenscavenging agents, for sampling of both aerobic and facultative anaerobic organisms..

[Audio] 3) Classification based on functional use or application: Anaerobic media Anaerobic 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 media Thioglycollate medium.

. . Lid with O-ring gasket Envelope containing sodium bicarbonate and sodium borohydride Anaerobic indicator (methylene blue) Petri plates Clamp with clamp screw C02 Palladium catalyst pellets.

[Audio] Media for Biochemical Identification TSI Urease Simmon's Citrate SIM.

. . Grarm Negative Biochenücal Chart Gran• Neg Bacteria E. coli E n terobacter Enterobacter Aerotnonas h •dro Klebsiella neutnonia Klebsiella oxytoca Citrobacter Proteus vulgaris Proteus rnirabilus Morganella Inoroanil Pseud ontonas Pseudoruonas titida Acinetobacter xv ffi• Providencia r ttoerl Shewanella u trefaci Sal Dliitnuriutn a tnone a -nnaænæ 2 K K K K.

[Audio] E. Coli Proteus mirabilis. . . E. Coli. Proteus mirabilis.

[Audio] Providencia rettgeri Morganella morganii.

[Audio] Psedomonas aeroginosa Klebsiella oxytoca.

[Audio] Mueller-Hinton agar it is a non-selective, non-differential medium it contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics it 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..

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[Audio] Quality tests chemical and biological parameter checked to ensure end products meet product quality specification, packaging, labelling and storage are important.

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