CULTURAL CHARACTERISTICS OF BACTERIA

Identification of bacteria - cultural, - biochemical - genetical characteristics. The cultural characteristics of an organism pertain to its macroscopic appearance on different kinds of media. - nutrient agar plates - nutrient agar slants - nutrient broth - nutrient gelatin

NUTRIENT AGAR SLANT CULTURE Inoculation: Agar slant is inoculated using an agar stroke.

Evaluation: After 48 hours at 37C, (a) Amount of growth: none, slight, moderate and abundant (b) Color: any pigmentation? (c) Opacity: transparent, translucent (partially transparent) and opaque. (d) Form:

Filiform: uniform growth along the line of inoculation. Echinulate: margins of growth exhibit toothed appearance. Beaded: separate or semiconfluent colonies along the line of inoculation. Effuse: growth is thin, unusually spreading. Arborescent: branched, treelike growth Rhizoid: root-like appearance.

NUTRIENT BROTH CULTURE

Evaluation: After 48 hours at 37C, gently (with no agitation) (a) Surface:

(b) Subsurface: turbid; granular; flocculent; flaky. (c) Sediment: agitate the tube, suspending the material. The type of sediment can be described as turbid, granular, flocculent, flaky and viscid. (d) Amount of growth: To determine the amount of growth, it is necessary to shake the tube to disperse the organisms. Terms such as; slight, moderate and abundant adequately describe the amount.

NUTRIENT GELATIN STAB CULTURE

Inoculation: Using aseptic techniques, inoculate each tube by inserting the needle into the center of the medium and continuing down the stab until the tip of the needle is within inch of the bottom of the tube. Carefully withdraw the needle, avoiding any lateral motion. Nutrient gelatin stabs will be incubated at 20C.

Evaluation: After 1 week at 20C, (a) type of growth and (b) presence or absence of liquefaction. (a) Type of growth (no liquefaction):

(b) Liquefaction: Some organisms elaborate the exoenzyme gelatinase, which is capable of digesting gelatin, a protein derived from collagen. gelatin polypeptides amino acids (transported into the bacterial cells)

Crateriform: saucer-shaped liquefaction Napiform: turnip-like liquefaction Infundibule: funnel-like or inverted cone-like Saccate: elongated sac, tubular, cylindrical Stratiform: liquefied to the walls of the tube in the upper regions.

NUTRIENT AGAR PLATE CULTURE

Evaluation: -color -opacity -form

-elevation

-margin

Certain risks when a culture is examined (i) unwanted organisms (contamination) (ii) resemblance of organisms (iii) species variation due to - an environmentally induced change of a genetically controlled characteristic: temporary - spontaneous and undirected permanent genetic changes, known as mutations: permanent - the acquiring of a new assortment of genes as a consequence of conjugation: permanent

GROWTH DEPENDENT IDENTIFICATION METHODS

(GROWTH ON SELECTIVE AND DIFFERENTIAL MEDIA)

A SELECTIVE medium is one to which compounds have been added to selectively inhibit the growth of certain microorganisms but not others. Selective media can be inhibitory or enrichment medium. A DIFFERENTIAL medium is one to which some sort of indicator, usually a dye, has been added, which allows to differentiate between various chemical reactions carried out during growth.

Some media used in growth dependent identification methods: Blood Agar (BA), Eosin Methylene Blue Agar (EMB), MacConkey Agar (MC), Salmonella Shigella Agar (SS), Xylose Lysine Deoxycholate Agar (XLD), Triple Sugar Iron Agar (TSIA), Bismuth sulphite Agar (BSA), DNase Test Agar, Hektoen Enteric Agar (HE), Mannitol Salt Agar (MSA),

Blood Agar (BA) -enriched medium for growing fastidious bacteria and differential medium. -Exotoxins called HEMOLYSINS cause lysis of the red blood cells. 1. Beta hemolysins completely lyse the red blood cells (RBC) and hemoglobin 2. Alpha hemolysis refers to the partial lysis of RBCs and hemoglobin (peroxide oxidizes hemoglobin to methemoglobin) 3. Gamma hemolysis results in no change of the medium. When reading plates, be sure to look for color changes in the surrounding agar. A common mistake is to look at the color of the bacteria, rather than the agar. Staphylococcus aureus shows beta hemolysis on BA.

Eosin Methylene Blue Agar (EMB) -selective and differential -for isolation and differentiation among members of the Enterobacteriaceae. -methylene blue, eosin dyes and lactose. -selects Gram negative bacteria, and differentiates those which ferment lactose. -small amounts of acid production result in a pink colored growth, while large amounts of acid cause the acid to precipitate on the colony, resulting in a characteristic greenish, metallic sheen. Organisms which do not ferment lactose will be colorless, taking on the color of the medium.

MacConkey Agar (MC) -selective AND differential -bile salts and crystal violet -inhibiting the growth of most Gr(+) bacteria. -Lactose and NEUTRAL RED -differentiates between lactose-fermenting coliforms and lactose nonfermenters, (potential pathogens).

Salmonella Shigella Agar (SS) -selective and diferential -Brilliant green, bile salts, sodium thiosulfate and citrate -inhibit all gr(+) and many gr (-) bacteria. -permit Salmonella and Shigella from environmental and clinical specimens -Lactose is the sole carbohydrate and NEUTRAL RED is the indicator -Sodium thiosulfate and Ferric citrate allow the detection of H2S producing bacteria, such as Proteus and some strains of Salmonella, since they produce colonies with black centers and a clear halo. -presence of coliform bacteria -> degradation of lactose to acid with the pH indicator neutral red.

Microorganisms Shigella and some Salmonella species Proteus and most Salmonella species Escherichia coli

Appearance of Colonies Colourless, translucent Translucent with a black centre Pink to red

Group NO 1 2 3 4 5 6 Each group will have: 1.Nutrient agar slant 2.Nutrient broth 3.Nutrient gelatin 4.Nutrient agar plate 5.Blood Agar 6.EMB agar 7.SS agar 8.MC agar

Organism E. coli B. subtilis S. aureus S. anatum S. marcescens Enterobacter aerogenes

Report
Aim Results and discussion - Observe the colonies and growth characteristics of bacteria and evaluate
them according to your manual Biref information about the selective and differential media that you used in the lab and compare your results with the expected results based on your organism Possible experimental errors

References