Question

2. Describe the 3 physical factors that control growth that we tested in lab and what were the results. Which organism survived the most in each of these conditions? Why? 3. Name 3 biochemical tests that confirm that the bacteria isolated is Staphylococcus aureus. Kirby Beurer 4. Describe the test that you would use to determine whether the S. aureus that you identified above. is a MRSA. 5. Outline the procedure for Gram stain and negative stain. What would these stains determine? 6. Name a test, besides gram stain, that would determine whether a colony is Streptococcus or Staphylococcus. What would the results for each organism be?

don't need a answer to question 2

Answer 1

ANSWER 4)

Methicillin resistance in S. aureus is defined as an oxacillin minimum inhibitory concentration (MIC) of greater than or equal to 4 micrograms/mL. MRSA infection is one of the leading causes of hospital-acquired infections and is commonly associated with significant morbidity, mortality, length of stay, and cost burden. MRSA infections can be further divided into hospital-associated (HA-MRSA) infections and community-associated (CA-MRSA) infections. MRSA is resistant to entire classes of β-lactams including cephalosporins and carbapenems and has higher risk of development of resistance to quinolones, aminoglycosides, and macrolides .Methicillin resistance in S. aureus is mediated through an altered protein called low-affinity penicillin binding protein (PBP2a). PBP2a is encoded by mecA gene which is present in chromosomal mobile genetic element called Staphylococcal cassette chromosome mec (SCCmec). Thus one of the sensitive method to study detect the MRSA is through PCR ( POLYMERASE CHAIN REACTION ) specifc primer for mecA gene. Those S.aureus which are MRSA their genomic DNA will get amplified due to presence of mecA GENE. Other method such as cefoxitin disc diffusion and oxacillin broth microdilution methods for detection of MRSA can be use in taking presence of mecA gene as reference.

ANSWER 5)

The Gram stain, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content. These stain pink. The performance of the Gram Stain on any sample requires four basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram’s Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with safranin.

Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with safranin to counter-stain and let stand for 45 seconds.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
10. Blot dry the slide with bibulous paper.
11. View the smear using a light-microscope under oil-immersion.

ANSWER 6)

CATALASE TEST

This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2). It is used to differentiate those bacteria that produces an enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci. Normally 3% H2O2 is used for the routine culture while 15% H2O2 is used for detection of catalase in anaerobes.

Principle of Catalase Test

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old.

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism.