MORPHOLOGY OF BACTERIA COLONIES Introduction Bacteria grow tremendously fast when supplied with an abundance of nutrients.

. Different types of bacteria will produce different-looking colonies.

The characteristics of a colony (shape, size, pigmentation, etc.) are termed the colony morphology. Colony morphology is a way scientists can identify bacteria.

Bergey's Manual of Determinative Bacteriology Commonly termed Bergey's Manual Describes the majority of bacterial species identified by scientists so far. Provides descriptions for the colony morphologies of each bacterial species.

Basic Elements in Identifying Colonies

Form - What is the basic shape of the colony? For example, circular, filamentous, etc. Elevation - What is the cross sectional shape of the colony? Turn the Petri dish on end. Margin - What is the magnified shape of the edge of the colony? Surface - How does the surface of the colony appear? For example, smooth, glistening, rough, dull, rugose, etc. Opacity - For example, transparent (clear), opaque, translucent (almost clear, but distorted vision, like looking through frosted glass), iridescent (changing colors in reflected light), etc. Chromogenesis - For example, white, buff, red, purple, etc. Consistency

Odor

Butyrous (butter-like) Viscous or stringy (a portion of it may come off the agar surface with the transfer needle) Rubbery (whole colony comes off the agar surface with the transfer needle) Dry, brittle or powdery (colonies that break when touched by a needle) Sweet Putrefactive Fruity

What Can Grow on a Nutrient Agar Plate? Bacteria Each distinct circular colony should represent an individual bacterial cell or group that has divided repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible patch. Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in shape Bacillus subtilis Escherichia coli Proteus vulgaris Staphylococcus aureus Streptococcus pyrogenes Yeasts Yeast colonies generally look similar to bacterial colonies.

Some species, such as Candida, can grow as white patches with a glossy surface. Round Yeasts Candida albicans Pink Yeasts Molds are actually fungi, and they often appear whitish grey, with fuzzy edges. They usually turn into a different color, from the center outwards. Black Mold (Aspergillus nidulaus) Green Mold (Trichoderma harzianum)

Molds

Other Fungi

Agar Slant

Moss green colonies, a white cloud, or a ring of spores can be attributed to the growth of Aspergillus, which is common in such fungal infections as athlete's foot. Aspergillus

Agar butt Growth only within the line of inoculation (non-motile) Growth spread or not only within the line of inoculation (motile) Agar broth Amount (scanty, moderate, abundant) Distribution and type of growth Uniform (even turbid) Scum or film (pellicle) Sedimentary (granular) Ring at the top of the rim CULTURE MEDIA 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.

1. 2. 3.

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 (-) 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

bacteria.

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.

Enriched Medium organisms.

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). Broth or solid medium containing rich supply of special nutrients that promotes the growth of fastidious Prepared by adding extra nutrients to a medium called nutrient agar.

Blood Agar Types a) Blood agar plates (BAP) > Contains mammalian blood, typically at a concentration of 510%

>
b) >

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 C used for growing fastidious (fussy) respiratory bacteria, such as Haemophilus influenzae.

>
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

 a) b)
c)

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. Nutrient Agar Dissolve 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 plate Lay 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 slant Dispense 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 butt Dispense 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 1. petri dishes 2. test tubes 3. bunsen burners/alcohol lamps 4. wire inoculating loops 5. bottles of staining reagents 6. 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. 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 lamps 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. Withdraw the inoculating loop and immediately flame-sterilize it. Pass the mouth of the agar tube on the alcohol lamps 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.

Godspeed and Goodluck. ORA et LABORA

PowerPoint Presentation by Frances Rowena Mercado, MAED General Science Handout Transferred by: em looney of BSN II 3