bacterial cell culture(media preparation)

8/9/2019 Bacterial Cell Culture(Media Preparation)

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BACTER45P- Media Preparation


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Diagnosis of Disease Initial step in studying morphology and

identification Obtain Antigen from developing serological

assays or vaccines Genetic Studies Reliable in estimating the number of viable

count Separate and isolate bacteria in mixtures


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The survival and growth ofmicroorganism depend on

available nutrients andfavorable growth

development

When culturingbacteria, it is veryimportant to provide

similar environmentaland nutritionalconditions that exists

in its natural habitat


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Artificial Culture Mediacontains:

Water Some of carbon energy Source of nitrogen Trace elements Growth factors Appropriate pH


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Water

Peptone Casein hydrolysate Meat extract Yeast extract Malt extract


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Consistency Nutritional

component Functional use


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A. Liquid mediAB. Solid MediaC. Semi Solid MediaD. Biphasic Media


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Used to propagate large numbers of

microorganisms in fermentation studies andvarious chemical tests

Suitable to grow bacteria when the numbersin the inoculum is suspected to be low

Used when a large number of bacteria haveto be grown


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Inoculating in the liquid medium also helps to

dilute any inhibitors of bacterial growth Used to obtain viable count( dilution method) Presence of more than one type of bacteria

can not be detected.


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Example:


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Supports growth of all types of organism Resazurin- indicator of anaerobicity O.075% agar prevent convection current From carrying atmospheric oxygen through out

the broth

Thioglycolic Acid( Reducing Agent) create ananaerobic environment deeper in the tube.Thusallowing anaerobic bacteria to grow.


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1.If only the top has growth, that means it is a strict aerobe because it is growing only in the

top part.

2. If the bottom has growth, it means it’s a strict anaerobe (aka obligate anaerobe) because it

can grow in the portion furthest away from the oxygen.

3. If there is growth throughout, but mostly at the top, it is a facultative bacteria since it

likes aerobic respiration best but can grow without it as well.

4. If there is only growth just below the very top, that’s indicative of a microaerophile, because it

needs oxygen to live, but not a lot of oxygen, so it’s not going to grow closest to the top.

5. If there is growth throughout evenly dif fused, that’s indicative of an aerotolerant anaerobe as

the presence of oxygen doesn’t affect them.


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Any liquid media by the addition of certainsolidifying agents

Used:1. Surface growth of microorganism(colony

appearance2. Pure culture inoculations3. Storage of culture4. Biochemical reactions


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1-3 % agar concentration to make themedium solid

Agar is the most commonly used solidifyingagent.

Obtain from cell membranes of red

algae(Gelidium)

Long chains polysaccharide(70% agarose and 30%agaropectin)

Melts @ 95c and soilidifies @ 42c


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Example:


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Agar of 0.2- 0.5% renders the medium semisolid

Fairly soft Useful in demonstrating bacterial motility Motility is best observed at room

temperature Example: SIM, Stuart’s Amies, Cary Blair


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Culture system comprises of both liquid andsolid medium in the same bottle

Besides agar, egg yolk and serum can be usedto soilidify culture media

Serum and egg(normally liquid) can berendered solid by coagulation using heat

Example: Loeffler’s Serum Slope, LowensteinJensen Medium Dorset egg Medium-solidified as well as

disinfected by a process of inspissation


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Simple Synthetic

Complex Tissue


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Can support non fastidiousbacteria

Non fastidious- able to growwith minimal requirements

Peptone water and NA


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Exact composition is known Example: Davis and Mingioli Medium- Research

Purposes


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Has ingredients whose exact components aredifficult to estimate

Contain at least 1 component which is notchemically defined example: blood , serum,yeast


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For organism which cannot grow on cell freemedia

Chick Embryo

Mc Coy cells (mouse cells)-

Chlamydia


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Vero Cells-Derived from Africangreen Monkey Kidney Cells

A549 cells- Lung CarcinomaHela Cells- Cervical carcinoma

Hep2 Cells- Laryngeal Carcinoma


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Basal Media Enriched media

Enrichment media Selective Media


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Basically simple media that supports mostnon fastidious organism

Example:peptone water, NA


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contain nutrient supplement that supportsmost non fastidious bacteria

Addition of extra nutrients in the form ofblood, serum, egg yolk, etc to basal medium Used to grow nutrionally exacting (fastidiuos

bacteria) Example: BAP, CAP, LSS


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Liquid media that enhance the growth oforganism and serve to inhibit commensal in

the clinical specimen Example: Selenite broth and tetrathionate

broth for Salmonella and Shigella Alkaline peptone water- Vibrio


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select for growth of a partial law organism. - addition of certain inhibitory agents that

don’t affect the pathogen. Various approaches to make the medium

selective include the addition of antibiotics,dyes, chemicals, alteration of ph, or acombination of these.


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Inhibitors for Gram + bacteria1. Dyes : Gentian violet; crystal violet

2. Bile Salts: Sodium desoxychocolate

Inhibitors for Gram Negative Bacteria1. Na azide2. Potassium tellurite


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- Different bacteria can be recognized on thebasis of their colony color.

- various approaches include , incorporationof dyes, metabollic substrates so that thosebacteria that utilize them appeara asdifferently colored colonies.

E.g. MAC, TCBS


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Clinical specimen must be transported to thelaboratory immediately after collection to

prevent overgrowth of contaminatingorganisms or commensals Semi soilid media Prevent drying(dessication) of specimen Maintain the pathogen commensal ratio Inhibit overgrowth of unwanted bacteria


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Charcoal is added to

neutralize inhibitory factors

and to absorb fatty acidswhich are toxic to some

bacteria.


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Anaerobic bacteria need special media forgrowth because they need low oxygen

content and reduced oxidation reductionpotential and extra nutrients Boiling of media serves to expel any dissolved

oxygen 1% glucose, 0.1% thioglycollate, 0.1%

ascorbic acid, 0.05% cystein, reed hot ironflings make the medium reduced


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- a process of rendering a medium or materialfree of all forms of life.

3 methods: 1. autoclaving – 121c @ 15psi for 15 min 2.dry heat 160-170 for 2 hrs

-glasswares **3. Bacteriologic filter -more than 121 c

- >0.22um


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Ex. Scintered glass▪ Seitz asbestos pad filter

▪ Cellulose or polycarbonate base *

▪ Pls see safety consideration/precaution page 54.


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1. refrigerator to avoid dehydration 2.agar plates are air tight

3.tube media with fitted capped is store atroom temp4. agar/plate should be warmed before use


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Before autoclaving ,cover each tube withcotton

Place tubes in large beaker and cover withfoil.


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BAP * Add 100 ml of distilled water per flask

+4ml blood Good for 5 plates of 20ml/plate

MRVP (Broth) *add 75 ml distilled water and heat. Dispense 5 ml/tube. Autoclave


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SIM (butt) * add 75ml distilled water and heat.

Dispense 5ml/tube. Autoclave.

LIA (butt slant) * add 100 ml distilled water and

heat. Dispense 7 ml /tube. Autoclave


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TSI (butt slant) * add 100ml distilled water and

heat. Dispense 7ml/tube.Autoclave

SCI (slant) * add 75ml distilled water and heat. Dispense 5ml/tube. Autoclave


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BACTERIOLOGICAL MEDIABACTER45P

Common bacteriology media


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▪ Thayer Martin Vancomycin- inhibit gram positive bacteria

Colistin- inhibit gram negative bacteria except Neisseria

Nystatin- fungi inhibitor

▪ Modified Thayer Martin

Thayer Martin plus Trimethoprim lactate which prevent theswarming of Proteus

▪ Martin Lewis

Vancomycin

Colistin

Trimethoprim lactate

Anisomycin- inhibit the growth of fungi

▪ New York City Agar

Vancomycin+ Colistin+ Trimethoprim lactate and Amphotericin B

that inihibit the growth of fungi


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Medium: Mannitol Salt Agar( Selective and Differential) Inhibitor: 7.5 % NACl

CHO: Mannitol

pH indicator: Phenol Red (Acid= Yellow; Alkaline= Red)

Mannitol Fermenter- Yellow colony due to production of acid. Example: S. aureus

Non Mannitol Fermenter- Pink- S. epidermidis and

S. saprophyticus


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Medium: TCBS( Thiosulfate Citrate Bile Salts) CHO: Sucrose

pH indicator: Bromthymol blue

Acid: Yellow; Alkaline: Yellow

Sucrose fermenter- Vibrio cholerae; Vibrio alginolyticus Non sucrose fermenter- Vibrio parahemolyticus- green

colony


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Rapid LactoseFermenters• Escherichia• Klebsiella• Enterobacter

Late Lactosefermenter•

S. arizoane• S. sonnei• Serratia• Hafnia• Yersinia

Non LactoseFermenter• Salmonella• Shigella except S.

sonnei

• Proteus• Providencia• Morganella• Edwardshiella•

Erwinia-plantpathogen


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• Selective and differential Media• CHO: Lactose• Lactose fermenter: Pink to Purple

Colonies• Non Lactose fermenter: Colorless

Colony• E.coli- Green Metallic Sheen• Klebsiella- Pink Mucoid colonies• Enterobacter- Pink colonies with dark

center” FISH EYE”

Eosin Methylene Blue


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•Purpose: Blood agar is used to determine the ability of anorganism to hemolyze erythrocytes (RBCs).

• Medical Applications The ability to produce hemolysis isassociated with virulence. Many pathogenic species ofStreptococcus, Staphylococcus and Clostridium demonstratehemolysis.

• Streptococcus pyogenes, which causes streptococcal sorethroat, scarlet fever, rheumatic fever, and other diseases, is (βhemolytic.

• S. pneumoniae, which causes pneumonia and pneumococcalmeningitis, is a hemolytic.

• Most strains of Staphylococcus aureus, the causative agent of

boils, toxic shock syndrome, food poisoning and otherdiseases, are β hemolytic.• Clostridium botulinum and C. tetani are β hemolytic and cause

botulism and tetanus, respectively.


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• Principle Exotoxins that cause destruction of RBCs (hemolysis) arecalled hemolysins.

• There are three categories of hemolysis.• β hemolysis is the complete destruction of RBCs and hemoglobin and

results in a clearing around the growth on a blood agar plate.•

Partial destruction of RBCs and hemoglobin (α hemolysis) produces agreenish discoloration of the blood agar plate.• In γ hemolysis, there is no destruction of RBCs and hemoglobin, so

there is no change in the medium.• Alpha-prime-hemolysis (α′ ) – A small zone of complete hydrolysis

that is surrounded by an area of partial hemolysis


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“What is

essential isinvisible to

the nakedeye”

bacterial cell culture(media preparation)

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