ID: 809001347 TITLE: Biochemical tests-gram positive bacteria METHODOLOGY: as given in lab guide OBSERVATIONS: TABLE 1 SHOWING MACROSCOPIC

AND MICROSCOPIC MORPHOLOGY OF COLONIES FROM PLATES A AND B.

PLATE

# OF
COLONIES

MACROSCOPIC MORPHOLOGY OF COLONY (24hrs) - Multiple punctiform, circular, opaque colonies, mucoid in texture and raised off the agar with entire margins. It was present in the 1st-5th quadrant, with single colonies in quadrant four and five. -Translucent boarder around colonies were present - Multiple punctiform, circular opaque and mucoid/buttery colonies. It was raised off agar with entire margins. Was present in all five quadrants. Single colonies were present in the fourth and fifth quadrants

MACROSCOPIC MORPHOLOGY OF COLONY (48hrs) -Colonies appeared the same with the exception of incomplete haemolysis (greenish to brown boarder around colonies)

MICROSCOPIC MORPHOLOGY OF COLONY Gram positive, cocci (purple) Branching chains, clusters

-colonies appeared the same with the exception of complete haemolysis (transparent boarder appeared around the colonies)

Gram positive, cocci (purple) Clusters, tetrad, paired and single cells

TABLE 2 SHOWING RESULTS FROM CATALASE AND COAGULASE TESTS CULTURE STAPH. AUREUS +VE CATALASE TEST +VE/-VE OBSERVATIONS Bubble formation upon addition of colony Bubble formation upon addition of colony Bubble formation upon addition of colony Bubble formation upon addition of colony +VE COAGULASE TEST +VE/-VE OBSERVATIONS milky initially then became transparent with clumps Appeared clear

STAPH. EPIDERMIDIS CULTURE A

+VE

-VE

+VE

-VE

Appeared clear

CULTURE B

+VE

+VE

milky initially then became transparent with clumps

DISCUSSION: Bacteria can be divided into two groups based on their staining capabilities when undergoing gram staining. That is based on their chemical and physical properties of the cell wall; bacteria are able to retain the crystal violet stain or not. Gram positive bacteria are able to retain the crystal violet characteristic purple color due to the thick peptidoglycan layer in the cell wall. The crystal violet ions are able to attach to the negative charged cell wall of the bacteria. The iodine added is used to increase the affinity of the crystal violet to the cell wall by binding to the crystal violet and making it non disolvable. The decolorizer (alcohol) is used to remove the crystal violet iodine complex. The safronin counter stain is used to re-stain the bacteria; however the crystal violet color masks the color of the safrinin in gram positive bacteria. Pink cocci may be seen on the slide due to over decolorization by the alcohol (decolorizer). Plate A appeared to have under gone incomplete haemolysis due to the appearance of the greenish boarder around the colonies. After 48hrs the bacteria was able to reduce the red blood cells hemoglobin in the blood agar media. This is referred to as partial / beta haemolysis since the cell wall of the red blood cells is still intact. For the Staphylococcus bacteria, a double haemolysis is supposed to appear with both alpha and beta haemolysis, however beta haemolysis was more visible in plate A. For plate B, alpha haemolysis was visible since a transparent boarder was visible around the colonies. This occurred because of complete lyses of the red blood cells in the blood agar media. Staphylococcus epidermidis however is not supposed to undergo haemolysis hence the recording could be of some other reasoning. The catalase test is done to differentiate between aerobic and anaerobic bacteria. The principle is that catalase (enzyme in bacteria) is able to convert hydrogen peroxide into water and oxygen as a defense mechanism against oxidizing agents which destroy cellular contents. Obligate aerobes and facultative anaerobes contain such an enzyme and as result of adding the colony to the hydrogen peroxide, effervescence / bubbles were seen due to the release of oxygen from the chemical reaction. A possible false positive result can occur if the procedure is done reversed or red blood cells from the media added also. So from the results, culture A,B, Staph. aureus and epidermidis) are either obligate aerobes or facultative anaerobes. The principle behind the coagulase test is the fact that coagulase (enzymes) are capable of clotting blood plasma by a mechanism similar to that of normal clotting. The process involves the conversion of fibrinogen into fibrin. For bacteria this process is a virulence factor since the fibrin forms a meshwork around the bacteria, protecting it from the immune response of the host (phagocytosis). The test can be used to classify bacteria into virulent and non virulent categories. Hence from the results: Staphyoloccus aureus and culture B are more virulent and pathogenic than the Staphylococcus epidermidis and culture A. Hence in conclusion we can assume from the results that culture B contains Staphylococcus aureus while culture A contains Staphylococcus epidermidis. Hence staphylococcus aureus are facultative anaerobe/ obligate aerobes and virulent, gram positive, hemolytic, catalase and coagulase positive bacteria. Staphylococcus epidermidis are facultative anaerobe/ obligate aerobes, gram positive, hemolytic, catalase positive but coagulase negative; with no virulence. One further test that can be done to differentiate between the species of Staphylococcus is the mannitol salt sugar. It is a selective and differential test where the strains of Staphylococcus are streaked and inoculated on the media. Organisms such as Staphylococcus aureus that are capable of using mannitol as a food source will produce an acidic product which changes the pH of the media. As a result, the pH indicator (phenol red) will turn yellow. Staphylococcus epidermidis lacks such as characteristic and hence the culture will not change color.