Dr. Hashem Ahmed

NO FOOD OR DRINK ALLOWED IN THE LABORATORY

NO MOUTH PIPETTING

INDIVIDUALS NOT ALLOWED TO WORK IN THE LABORATORY

ALONE

WEAR REQUIRED PERSONAL PROTECTIVE EQUIPMENT

NO SMOKING IN LABORATORY

MAINTAIN GOOD HOUSEKEEPING HABITS

Remember

Microbial cultureCulture mediaCulture conditionsMeasurement of growth

MediaIn order to recover or isolate bacteria for

study, you need to provide all of the growth demands and growth conditions.

Combination of all of the ingredients that supports the growth of microorganisms is called media

Types of mediaDifferent approaches for classifications of

types of media used in microbiology are available.

Culture Conditions

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1. Obligate Aerobes

2. Obligate Anaerobes

3. Facultative Aerobes , Facultative Anaerobes

4. Microaerophilic

Culture Media

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Culture MediaA. compositions 1. Chemically defined

The exact chemical composition is knownUsed to grow fastidious organisms

2. Complex mediaExact chemical composition is not knownMost bacteria and fungi are grown with this

Culture MediaB. consistency 1. Broth

Media provided in liquid phaseused to measure motility, aeration and

counting of bacteria2. solid Media

Media with solidifying agents like agarUsed for recovery of bacterial colonies

Agar : Solid medium is made by adding Agar Agar is obtained from Sea weeds Agar contains long chain polysaccharides.

Inorganic salts and protein like substancesIt melts at 980c and sets at 420c It is not degraded or metabolized by

bacteria

Liquid media can be converted to solid media by adding 1-2% of agar

If you add 0.6% agar, the media is called semisolid

Petri Dish With Media :

Petri dish with Media Plate: provide large surface for isolation and observation of colonies

Using a sterile loop or a sterile swab streak your sample on the petri plate

Important let your sterilized loop cool before you pick up your sample

Culture mediaC. Purpose and use

1. General purpose media Media provided in liquid phase or agar to provide the

minimal and basic growth requirementsNot all microbes can grow on such mediaIt is a complex, general purpose mediumExamples:• Nutrient agar,• Trypticase soy broth ((TSB, or tryptone soya agar

(TSA)): is frequently the base media of other agar plate type, i.e. blood agar plates (BAP) are made by enriching TSA plates with blood

• Mueller Hinton agar: commonly used for antibiotic susceptibility testing, it contains starch (Starch is known to absorb toxins released from bacteria, to prevent interfere with the antibiotics. Second, it is a loose agar. This allows for better diffusion of the antibiotics. BETTER DIFFUSION = TRUER ZONE OF INHIBITION.

2. Enriched mediaIs a medium with specific and known qualities that favors the growth of a particular microorganism. Media provided with additional growth requirements to enable the growth of fastidious microorganisms(by adding blood, serum or egg).

Best obtained by additions of 5% sheep or horse blood Examples:• Blood agar•Selenite broth is used to selectively isolate Salmonella species.• Alkaline peptone water is used for the cultivation of vibrio.

3. Selective Media Inhibits the growth of some bacteria while

selecting for the growth of othersExample:

Brilliant Green Agar, MacConky or CLED (Cystine Lactose Electrolyte Deficient Media))

Dyes Inhibit The Growth Of Gram (+) Bacteria Selects For Gram (-) Bacteria Most G.I. Tract infections are caused by Gram (-)

bacteria

Selective Media

EMB (Eosin Methylene Blue)Dyes inhibit Gram (+) bacteriaSelects for Gram (-) bacteriaG.I. Tract infections caused by Gram (-)

bacteria

Selective Media

MacConky agarDyes inhibit Gram (+) bacteriaDelects for Gram (-) bacteria by bile saltsG.I. Tract infections caused by Gram (-)

bacteria

4. differential media Differentiates between different organisms

growing on the same plateExample:

Blood Agar Plates (TSA with 5% sheep blood) Used to differentiate different types of

StreptococciMacConky agar

Used to differentiate between lactose and non lactose fermenting bacteria

Alpha Hemolytic Streptococci

Incomplete lysis of RBC’s

Beta Hemolytic Streptococci

Complete lysis of RBC’s

Selective and Differential MediaMannitol Salt Agar

Used to identify Staphylococcus aureusMannitol Salt Agar

High salt conc. (7.5%) inhibits most bacteria

Sugar MannitolpH Indicator (Turns Yellow when acid)

Selective and Differential MediaMacConkey’s Agar

Used to identify G-veMacConkey’s Agar

Bile salts and crystal violet (inhibits Gram (+) bacteria)

lactosepH Indicator

Many Gram (-) enteric non-pathogenic bacteria can ferment lactose, Salmonella can not

Special Culture Techniques1. Anaerobic Bacteria

a. Reducing Mediab. Anaerobic Containerc. Agar Stab: stab culture  one in which the

medium is inoculated by thrusting a needle deep into its substance.

d. Agar Shake: shake culture a culture made by inoculating warm liquid agar culture medium in a tube and shaking to distribute contents evenly. Incubation of the resolidified culture allows the development of separated colonies; especially adaptable to obligate anaerobes.

Special Culture Techniques2. Microaerophilic Bacteria

Grow best under reduced O2 levels and increased CO2 levels

Normal Atmosphere 21 % O2

3 to .03 % CO2

Microaerophilic Bacteria

A. Candle Jar

16 % O2

4 % CO2

Microaerophilic Bacteria

B. CO2 Generating Packet

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The End