Bacterial culture media بكتريا عملي

Bacterial Culture Medium
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University

Culture and Medium
•Culture is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them.
•Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth.
- Nutrients (agar, pH indicators, proteins and
carbohydrates) used to grow organisms
outside of their natural habitats
Dr. Nabil El Aila General Microbiology

Basic requirements of culture media
•Nutrients - Energy source - Carbon source - Nitrogen source
•Mineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca.
•A suitable pH – 7.2 – 7.4
•Accessory growth factors - Tryptophan for Salmonella typhi - X & V factors for H. influenzae

Agar - Agar
•Agar is obtained from Sea weeds
•Agar contain long chain polysaccharides. Inorganic salts and protein like substance
•Used as solidifying agent for culture media in Petri plates, slants, and deeps
•Generally not metabolized by microbes
•Liquefies at 98 C and Solidifies ~42 C

5
 Culture medium
–is the mixture of various nutrients that is suitable for the growth of microorganisms.
 Types of Culture Media based on the function and chemical components based on the physical state

6
Based on the physical state
•Liquid medium:
–Without agar.
–for the proliferation of bacteria.
•Solid medium:
–1.5-2.5% agar.
–for the isolation and identification of bacteria
–e.g., slant, Petri dishes/plates.
•Semisolid medium:
–0.3-0.5% agar.
–for the observation of bacterial motility and preservation of bacteria.

ii.Growth of bacteria in culture medium
i.Liquid medium or Broth
1)Homogeneous turbidity
2)Surface
3)Bottom
ii.Solid agar medium
Colony and mossy
1)Smooth colony
2)Rough colony
3)Mucoid colony
iii.Semi-solid agar medium

Blood culture – ‘Liquid Medium’

Semisolid medium
The organism in the tube on the left in the figure is motile and the organism in the tube on the right is nonmotile.
Dr. Nabil El Aila
General Microbiology

Types of culture media
•Basic media
•Enriched media
•Selective and enrichment media
•Enrichment media
•Differential media or indicator media
•Transport media
•Anaerobic media

•Basic media are basically simple media that supports most non-fastidious bacteria.
Peptone water, nutrient broth and nutrient agar considered
basic medium.

Peptone
•Peptone contain partially digested proteins
•Proteases
•Polypeptides
•Aminoacids
•Inorganic salts
Phosphates
Potassium and Magnesium
Riboflavin
Meat exract called as Lab lemco

Carbohydrate media
•Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% soln(1ml)
•Dissolve the desired carbohydrate in peptone water and steam for 30 min or sterilize by filtration.
•Use: To test the fermenting ability of an organism

Nutrient Agar
•Contain 2% agar added to Nutrient agar commonly used
•Concentration can be increased to 6% to prevent swarming
•Can be reduced to 0’5%

Pigment producing Staphylococci

•Enriched media: Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media.
- Enriched media are used to grow nutritionally exacting
(fastidious) bacteria.
- Blood agar, chocolate agar, Loeffler’s serum slope etc are
few of the enriched media.

Enriched Medium
•To culture medium Blood serum or egg are added to medium
• eg Blood agar
•Chocolate agar
•Egg

Different types of hemolysis on Blood Agar

Other Enrichments – Chocolate Agar
•Several organic materials are added to the basic constituents of the Medium such as Blood, yeast, yeast extract etc

•Basic media
•Enriched media
•Selective and enrichment media
•Enrichment media
•Differential media or indicator media
•Transport media
•Anaerobic media

•Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria.
- While selective media are agar based, enrichment media are
liquid in consistency. Both these media serve the same purpose.
- Any agar media can be made selective by addition of certain
inhibitory agents that don’t affect the pathogen.
- Various approaches to make a medium selective include
addition of antibiotics, dyes, chemicals, alteration of pH or
a combination of these.
- Examples of selective media like Salmonella shigella agar,
XLD agar, Heckton Enteric agar, Mannitol salt agar, Lowenstein
Jensen’s medium
•Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen.
- Selenite F broth, tetrathionate broth and alkaline peptone
water are used to recover pathogens from fecal specimens.

Salmonella Shigella agar
- For isolation and differential medium for pathogenic Gram-negative bacilli in particular, Salmonella and Shigella. Inhibitor for Coliforms.

For isolation of Salmonella and Shigella species from clinical samples
XLD agar (Xylose Lysine Deoxycholate)

For isolation of Salmonella and Shigella species from clinical samples
Hecktoen Enteric agar
Dr. Nabil El Aila

Lowenstein Jensen Medium - cultivation of Mycobacterium tuberculosis

Lowenstein-Jensen’s medium
•Mineral salt soln - 600ml Malachite green soln - 20ml (2gm% in D.water) Beaten egg - 1000ml (20-22 eggs)
•Mix the above
•Distribute in Mc Cartney bottles
•Sterilize by Inspissation
Use: To cultivate Mycobacteria

• For selective isolation of pathogenic staphylococci
• Mannitol fermentation by pathogenic staphylococci is indicated by a yellow halo surrounding the colonies.
•Sodium chloride is the inhibitor agent.
•Phenol red is the PH indicator
Mannitol Salt Agar ( MSA )

Enrichment Medium
•If the sample contain more than one type of bacteria, undesired bacteria grwoth can be reduced or eliminated.
•The desired organism is facilitated to grow
•Eg Tetrathionate broth
•Selenite F broth

•Differential media or indicator 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.
- Such media are called differential media or indicator
media.
- Examples: MacConkey’s agar, CLED agar, TCBS
agar etc.

MacConkey agar
•MacConkey agar is useful medium for cultivation of enterobacteria
•It contains a bile salt to inhibit non intestinal bacteria
•Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting Salmonella and Dysentery group

Differential Medium Mac Conkey's agar
•Bringing out different characters of bacteria their atypical characters
•Mac Conkey’s medium
Contain peptone, Lactose Agar, Neutral red and taurocholate and show growth of Lactose fermenters as pink colored colonies

Lactose fermenting and Non lactose fermenting

TCBS medium (Thiosulphate Citrate bile salts sucrose agar) For cultivation of Vibrio cholera

Cystine-Lactose-Electrolyte-Deficient Agar (CLED agar) For isolation and differentiation of urinary pathogens on the basis of lactose fermentation

•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.
- Such media prevent drying (desiccation) of specimen, maintain the
pathogen to commensal ratio and inhibit overgrowth of unwanted
bacteria.
- Some of these media (Stuart’s & Amie’s) are semi-solid in
consistency.
- Addition of charcoal serves to neutralize inhibitory factors.
- Cary Blair medium and Venkatraman Ramakrishnan medium are
used to transport feces from suspected cholera patients.
- Sach’s buffered glycerol saline is used to transport feces from
patients suspected to be suffering from bacillary dysentery.

Transport Medium
•Stuart’s medium contain reducing agents to prevent oxidation.
•Charcoal to neutralize certain bacterial inhibitors to Gonococci,
Dr. Nabil El Aila

•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.
•Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced.
•Robertson cooked meat that is commonly used to grow Clostridium spps.
•Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the thioglycollate medium. Under reduced condition, methylene blue is colorless.

Anaerobic Medium
•Robertson’s cooked meat medium
•Thiglyclolate liquid medium

Dr. Nabil El Aila

Anaerobic Jar (GasPak)
Clamp with clamp screw
Lid with O-ring gasket
Catalyst pellets in reaction chamber
Flash arrester to
prevent explosion
GasPak disposable anaerobic indicator strip
Culture plates
GasPak disposable
hydrogen and carbon
dioxide generator
envelope
O
O
O
H
H
H
H
Oxygen in jar
(O2)
Water (H2O)
Hydrogen gas (H2)
+
2
2
Platinum catalyst

Other/ungrouped media

Mueller Hinton Agar
•It is a microbiological growth medium that support the growth of most microorganism.
•It is commonly used for antibiotic susceptibility testing
•typically contains (w/v):
- 30.0% beef infusion
- 1.75% casein hydrolysate
- 0.15% starch
- 1.7% agar
•pH adjusted to neutral at 25 C.
•Five percent sheep blood may also be added when susceptibility testing is done on Streptococcus species or the
sensitivity can be done on Blood agar.

Muller Hinton Agar for Antibiotic Testing
Dr. Nabil El Aila

Antibiotic Testing on Blood Agar Medium

Triple Sugar Iron Agar (TSI):
•It is used for identification of a Gram- negative bacilli of the enteric group.
•It contains glucose (0.1% ), Lactose (1%), sucrose(1%). And peptone (2%) as nutritional sources.
•Sodium Thiosulfate serves as the electron receptor for reduction of sulfur and production of H2S.
•Detects fermentation of sucrose, lactose, glucose, as well as production of hydrogen sulfide and /or gas .
•Phenol red is the PH indicator; ferric ammonium citrate is H2S indicator.

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Triple sugar iron slant
Dr. Nabil El Aila

The lysine iron agar slant or LIA slant test is used to distinguish bacteria which are able to decarboxylate lysine and/or produce hydrogen sulfide from those that cannot. This test is particularly useful for distinguishing different Gram-negative bacilli—especially among the Enterobacteriaceae
Lysine Iron Agar (LIA):

Simmons' Citrate Agar is a defined, enrichment medium that tests for an organism's ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source.
Simmons' citrate agar

Urea agar base
Urea Agar Base (Christensen Agar Base). for the detection of urease producing organisms.

Sabouraud's Dextrose agar commonly used Fungal Isolation Medium

Sabouraud's Dextrose Agar
Dextrose - 4 gm%
Neopeptone - 1 gm%
Agar - 1.5 gm%
Distilled water - 100 ml
•Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4
•Autoclave and dispense 20 ml amount in test tubes
Use: For the cultivation of Fungi
Dr. Nabil El Aila

Sterilization of culture media
•Media are sterilized in the autoclave at 1210 c for 15
min under 15 lb of Pressure
•Heat-labile substances like serum & sugar solutions must be sterilized by free-steam or filtration
•Egg containing media –-- Lowenstein-Jensen’s medium, Loeffler's serum slope by inspissation
•Discarded culture plates are to be sterilized by autoclaving prior to washing
Dr. Nabil El Aila

Storage of culture media
•Prepared media in individual screw capped bottles can be stored for weeks at room temp
•Poured plates deteriorate quickly and often contaminated, hence cold storage is necessary
•For smaller labs domestic refrigerators & for larger labs insulated cold room(4-5oc)
•Deep freeze refrigerators for preservation of sera, antibiotics & amino acids (-10 to - 400c)

Bacterial culture media بكتريا عملي

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