tuba bashir

CCULTURE MEDIA ULTURE MEDIA USED IN USED IN

MICROBIOLOGYMICROBIOLOGY

Tuba BashirSupervisor Microbiology Department

THE INDUS HOSPITAL, KARACHI

Story Behind Penicillin

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.

Definition, purpose/importanceHistory 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

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 them Bacteria have to be grown (cultured) for them to be identified and subsequent clinical to be identified and subsequent clinical diagnosis.diagnosis.By appropriate procedures they have to be By appropriate procedures they have to be grown separately (isolated) on culture media and grown separately (isolated) on culture media and obtained as pure for study.obtained as pure for study.Bacteria have to be cultured in order to obtain Bacteria have to be cultured in order to obtain antigens from developing serological assay for antigens from developing serological assay for vaccines.vaccines.Certain genetic studies and manipulations of Certain genetic studies and manipulations of the cells also need that bacteria be cultured in the cells also need that bacteria be cultured in vitro.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. It was on to grow bacteria. It was on the suggestion of the suggestion of Fannie EilshemiusFannie Eilshemius, , wife of wife of Walther Hesse Walther Hesse (who was an (who was an assistant to Robert Koch) that agar assistant to Robert Koch) that agar was used to solidify culture media.was used to solidify culture media.

History of culture medias

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

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 medium 2% agar is employed in solid medium

Agar Fibers Agar PowderAgar Fibers Agar Powder

Bacterial Growth Curve

Properties of MediaProperties 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: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 .

Classification:

Bacterial culture media can be classified in at least three ways

1.CONSISTANCY

2.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 An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients) used to culture (typically agar plus nutrients) used to culture microorganismsmicroorganismsAgar 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

e.g: SIM

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:Complex media:

specially prepared media for research purposes where the composition of every component is well known.

Synthetic media:Synthetic media:

3) Classification based on functional use or application:

Basal mediaEnriched mediaSelective media

Enrichment media Differential mediaTransport mediaAnaerobic media

Media for Biochemical Identification


• Basal mediaBasal media are basically simple media that supports most non-fastidious bacteria

Examples of Basal media:Examples of Basal media:Peptone water,nutrient broth and nutrient agar


Enriched mediaEnriched 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:Examples of Enriched media: Blood agarBlood agar Chocolate agarChocolate agar

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

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


Selective mediaAny 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

• LOWEINSTON JENSON (L.J )IS A SOLID MEDIUM USED FOR MTB

• Sabouraud Dextrose Agar (SDA)

• Thiosulfate-citrate-bile salts-sucrose agar (TCBS)


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.


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 mediaExample of differential mediaMacConkey’s 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

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

BIGGYBismuth 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


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 mediaExample of Transport mediaCary Blair medium for campylobacter species

Alkaline peptone water medium for V. cholerae

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 oxygen-

scavenging agents, for sampling of both aerobic and facultative anaerobic organisms.


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 Example of Anaerobic mediamediaThioglycollate medium

Media for Biochemical Identification

TSIUreaseSimmon’s CitrateSIM

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.

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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