CULTURE MEDIA

PowerPoint Presentation by Frances Rowena Mercado, MAED General Science

Culture Media

A liquid or gel designed to support the growth of microorganisms or cells.

Culture medium- nutrients prepared for microbial growth

Inoculation- introduction of microbes into medium

Culture/Colony- microbes growing in/on culture medium

In the history…

Robert Koch- described his culture techniques in 1881.

Fanny/Frau Hesse- suggested the use of agar. Richard Julius Petri- invented the glass Petri

dishes. Joseph Lister- the first person to obtain a pre

culture of bacterium (Streptococcus lactis) in a liquid medium.

Classification of Culture Media Based on Whether the Exact Contents are Known

Chemically defined media- exact chemical composition is known

Complex media- exact contents are not known, from extracts and digests of yeasts, meat, or plants

Liquid and Solid Media Liquid media- or broths are contained in tubes,

referred to as tubed media. Solid media- prepared by adding agar to liquid

media and then poured into test tubes or Petri dishes, where the media solidifies. Agar plate - one grown on a medium, usually agar or

gelatin, on a Petri dish Agar slant - one made on a slanting surface of a

solidified medium in a tube, the tube being tilted to provide a greater surface area for growth.

Agar butt/deep - one in which a tube of solid medium is inoculated by a needle thrust deep into the contents.

Bacterial Media

Selective Differential Enriched

Selective Medium

Has added inhibitors that discourage the growth of certain organisms without inhibiting growth of the organism being sought.

Solid medium is employed with selective medium so that individual colonies may be isolated.

Examples: MacConkey agar- screen for S. aureus and is selective

for Gram (-) bacteria. Phenylethyl alcohol agar (PEA) and colistin-nalidixic

acid agar (CNA)- inhibit growth of Gram (-) bacteria. Thayer-Martin agar and Martin-Lewis agar- selective

for N. gonorrhoeae. Mannitol salt agar (MSA)- only for salt-tolerant

(haloduric) bacteria Eosin methylene blue agar (EMB) – selective against

gram-positives

MacConkey agar E. coli on EMB

Differential Medium

Permits the differentiation of organisms that grow on the medium.

Reveals the presence of 2 or more similar microorganisms by differences in the appearance of their colonies.

Examples MacConkey agar- used to differentiate various

Gram (-) bacilli that are isolated from fecal spcimens. Gram (-) bacteria are able to ferment lactose

produces pink colonies, those are unable to ferment lactose produce colorless colonies.

Differentiates between LF and NLF Gram (-) bacteria. Mannitol salt agar- used to screen for S.

aureus, pink to yellow. Centrimide agar - used for the differentiation of

strains of Pseudomonas spp.

P. aeruginosa on centrimide agar Two different species of Staphylococcus growing on

mannitol salt agar (MSA).

Enriched Medium

Broth or solid medium containing rich supply of special nutrients that promotes the growth of fastidious organisms.

Prepared by adding extra nutrients to a medium called nutrient agar.

Blood Agar Types Blood agar plates (BAP)

Contains mammalian blood, typically at a concentration of 5–10%

Used to isolate fastidious organisms and detect hemolytic activity (Neisseria and Streptococcus).

Chocolate agar (CHOC)blood cells have been lysed by heating the cells

to 56 °Cused for growing fastidious (fussy) respiratory

bacteria, such as Haemophilus influenzae.

BAP CHOC

Remember…

Various categories of media are not mutually exclusive.

Ex: blood agar is enriched and differential MacConkey agar and MSA are selective and

differential PEA and CNA are enriched and selective Thayer-Martin and Martin-Lewis are highly enriched

and highly selective Thioglycollate broth (THIO) is a liquid medium that

supports the growth of all categories of bacteria.

Examples

Hektoen enteric agar (HEA) - selective and differential agar primarily used to recover Salmonella and Shigella from patient specimens

Salmonella-Shigella agar (SS) – selective and differential for Salmonella and Shigella

Bile Esculin Agar (BEA) is a selective differential agar used to isolate and identify members of the genus Enterococcus

Fungal Media Sabouraud agar

Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most bacteria; also contains the antibiotic gentamicin to specifically inhibit the growth of Gram-negative bacteria.

Hay infusion agar Specific for the culturing of slime molds

(though not technically fungi). Potato dextrose agar

PDA is used to culture of certain types of fungi.

Preparation of CM Nutrient Broth

Dissolve 8 g of NB powder in 1000 ml of distilled water in an Erlenmeyer flask. Mix and heat over the magnetic stirrer until the medium becomes clear or transparent.

Dispense 8 ml of the medium into sterile test tubes.

Immediately stopper the tubes completely.Sterilize in the autoclave at 15 psi, 121 C for 15

mins.

Preparation of CM

Nutrient AgarDissolve 28 g of NA powder in 1000 l of

distilled water in an Erlenmeyer flask. Mix and heat over the magnetic stirrer until the medium becomes clear or transparent.

Sterilize in the autoclave at 15 psi, 121 C for 15 mins.

Dispensing the CM

NA plateLay out several sterile Petri dishes on the table with

the cover partially open and dispense the medium inside and inoculating hood.

Dispense the NA medium (30 ml) and allow 5-10 minutes to elapse before completely covering the dish to avoid contamination.

When the medium is solidified, label the NA plate. Wrap and label the plate.

NA slantDispense 8 ml of the medium into sterile test

tubes using pipette or syringe.Cover the tube partially with sterile cotton plug

or screw cap and allow the agar medium to harden in an incline position on an agar slant rack.

Stopper the tube completely when the medium has solidified. Label the upper portion of the test tube slant.

Sterilize in the autoclave at 15 psi, 121 C for 15 mins.

NA deep or buttDispense 8 ml of the medium into sterile test

tubes using pipette or syringe.Cover the tube partially with sterile cotton plug

or screw cap and allow the agar medium to harden in an upright position on a test tube rack.

Stopper the tube completely when the medium has solidified. Label the upper portion of the test tube slant.

Sterilize in the autoclave at 15 psi, 121 C for 15 mins.

Inoculation of Culture Media

Inoculation- adding a portion of the specimen to the medium.

Involves the use of sterile inoculating loop to apply a portion of the specimen to the surface of the medium; a process commonly referred to as “streaking”.

Materials

petri dishes test tubes bunsen burners/alcohol lamps wire inoculating loops bottles of staining reagents incubators

Importance of Using “Sterile Technique”

Necessary to exclude all microorganisms from a particular area, so that area will be sterile.

Media should remain sterile before inoculation.

Contaminants- unwanted microorganisms Contaminated- if the sample contains

contaminants

Streaking the Agar Plate: Simple Streak

Flame sterilize the inoculating loop then let it cool for a few seconds, afterwards fish out a loopful of specimen from pure culture.

Hold the sterile Petri dish with cover by your left hand, partially open the agar plate near the flame of the alcohol lamp.

Place a loopful of specimen on one side of the agar medium away from you and streak the culture back and forth from edge to edge of the plate,

When the entire medium has been streaked, flame sterilized the cover of the plate completely.

Label the Petri dish then incubate for 24-48 hours at 37 C.

Streaking the Agar Plate

Inoculating the Agar Slant Fish out a loopful of bacterial culture using a

flamed sterilized loop. Hold the agar tube with the left hand and screw

cap should be held by the small finger. Never lay the screw cap of the test bacterial specimen anywhere.

Pass the mouth of the test tube through the alcohol lamp’s flame. Flame immediately and inoculate the agar surface by moving the inoculating loop from the bottom to the top of the agar slant or in a snake-like motion.

Inoculating the Agar Slant

Withdraw the inoculating loop and immediately flame-sterilize it. Pass the mouth of the agar tube on the alcohol lamp’s flame.

Sterilize the screw cap and cover the agar tube.

Label the agar slant tube and place it in the test tube rack, then incubate.

Inoculating the NB

Fish out a loopful of bacterial culture using a flamed sterilized loop.

Hold the agar tube with the left hand and screw cap should be held by the small finger. Never lay the screw cap of the test bacterial specimen anywhere.

Inoculate the medium from top to bottom. Assume equal distribution of inoculums by suspending the inoculating loop into the broth, then shake.

Flame sterilize the mouth of the test tubes before and after inoculation.

Sterilize the screw caps, cover, and label the broth culture, then incubate.

Inoculating the NA butt Fish out a loopful of bacterial culture using a flamed

sterilized loop. Hold the agar tube with the left hand and screw cap

should be held by the small finger. Never lay the screw cap of the test bacterial specimen anywhere.

Inoculate the inoculum into the agar butt by stabbing the inoculating needle into the agar without touching the sides of the test tubes at the middle and halfway of the agar.

Flame sterilize the mouth of the test tubes before and after inoculation.

Sterilize the screw caps, cover, and label the broth culture, then incubate.

FINISHED!