# Lab 6 Gram+ Bacteria: *Staphylococcus* and *Bacillus*

## BACKGROUND

Several Gram-positive bacterial species play critical roles in the food industry. In addition to LAB, the two examples we cover in this lab are *Staphylococcus aureus* and *Bacillus cereus*. While *Staphylococcus aureus* does not form spores, *Bacillus cereus* is a spore former. *Staphylococcus aureus* is carried on the body surface of most domestic animals and is part of the normal flora of the nasopharynx and skin of a large percentage of the human population. *Staphylococcus* can cause food poisoning by producing an extracellular enterotoxin. The toxin is thermostable and can cause symptoms of vomiting, diarrhea, and abdominal cramps. *Bacillus cereus* is mostly a soil microorganism. It can form spores, which is thought to be an adaptation to a feast-or-famine existence. The spores are tolerant to heat, desiccation, and a variety of toxic chemicals.

## OBJECTIVES

Be familiar with biochemical tests used for the differentiation and characterization of *Staphylococcus aureus* and *Bacillus cereus*.

## MATERIALS

### Cultures:
- *Staphylococcus aureus*
- *Staphylococcus epidermidis*
- *Escherichia coli*
- *Bacillus subtilis*
- *Bacillus cereus*

### Media:
- Baird-Parker (BP) agar
- Blood agar (BA, cow blood agar from the Vet Med.)
- Mannitol salt (MS) agar
- Vogel and Johnson (VJ) agar
- BHI broth
- Standard method broth + Bromcresol purple (SMBP) tubes
- Plate count agar (PCA)
- Fluid thioglycollate agar (FTA) tubes
- Skim milk agar (SKIM) agar
- Starch agar (SA) plates
- Triple sugar iron agar (TSI) slants
- Mannitol-Yolk-polymyxin agar (MYP) plate (check MYP agar in the media manual)
- MRVP broth

### Supplies:
- Lugol’s iodine
- Malachite green spore stain
- VP reagent
- 40% KOH
- Sterile mineral oil or vaspar
- Sterile saline
- Rabbit plasma for coagulase test
- Small test tubes for coagulase test
- Dri-block incubator for coagulase tests (37 ˚C incubator)
- Swabs
- Peroxide solution for catalase test

## PROCEDURES
### Procedures [VP for lab 6](https://drive.google.com/file/d/1Ns0i6MmkRliXHGwdAtvW8r4DG5srPSto/view?usp=drive_link);
[Additional growth media supplement information](https://drive.google.com/file/d/1-8g3r4xy-_JalJYOUZbOj7L8qPx_KwO4/view?usp=sharing)
### STEP 1: Microscopic Observation
1. Conduct a Gram stain for both cultures and record your observation.
2. Conduct endospore stain for the *Bacillus* strain and record your observation. The detailed protocol can be found [here](https://asm.org/ASM/media/Protocol-Images/Endospore-Stain-Protocol.pdf?ext=.pdf).
3. Make a wet mount of each organism and examine them under phase contrast microscopy for motility.

### STEP 2: Tests Associated with *Staphylococcus*
2.1 Inoculate the media for *Staphylococcus*
1. Inoculate the following media with the *Staphylococcus* strain: BP, BA, MS, VJ, BHI broth, SMBP, and PCA. Streak slants or plate and inoculate with a couple of loopfuls of liquid culture for broth media.
2. Incubate the inoculated media at 30 ˚C for 48 hours.

2.2 Are You a *Staphylococcus* Carrier?
1. Obtain one BP plate and divide it in half by using a marker and labeling the bottom of the plate.
2. Moisten two sterile swabs by dipping them into a tube of sterile saline and removing excess solution by gently squeezing the swabs against the inner wall of the tube.
3. For one swab, streak it directly onto one side of the BP agar plate.
4. Use the second swab to swab your nose and streak it on the other side of the BP agar.
5. Incubate the plate at 30 ˚C for 48 hours.

### STEP 3: Test Associated with *Bacillus*
3.1 Inoculate the media for *Bacillus*
1. Inoculate the following media with the *Bacillus* strain: PCA, FTA, SKIM, SA, BA, TSI, MYP, MRVP. Stab the FTA tube.
2. Incubate the inoculated media at 30 ˚C for 48 hours.

### STEP 4: *E. coli* Strain as a Gram- Control
Each group only needs to pick one *E. coli* strain and inoculate on one set of PCA, BA, MS, VJ, BHI broth, SMBP, FTA, SKIM, SA, TSI, MYP, and MRVP plates. Incubate at 30 ˚C for 48 hours.

### STEP 5: Read the *Staphylococcus* Results
1. Coagulase tests
1.1 Obtain coagulase plasma from the refrigerator.
1.2 Vortex the BHI tube and add 0.1 ml of culture taken from the BHI broth to the coagulase plasma in tube.
1.3 Incubate the tube at 37 ˚C for two hours and check every 30 min. Gently tilt the tube to check for the formation of a clot.
1.4 If the test is negative after two hours, incubate the test overnight at room temperature and check again tomorrow.
2. Check the color changes or other changes from the inoculated agar plate and record them.
3. Conduct a catalase test by mixing hydrogen peroxide with one colony picked from any of your agar plates. The production of bubbles indicates a positive test.
4. Don’t forget to check if you are a *Staphylococcus* carrier or not.

### STEP 6: Read the Results of the *Bacillus* Plates
1. Note the zones of clearing on the SKIM plate. Clearing of the agar indicates casein digestion.
2. Add several drops of Lugol’s iodine to the starch agar plate. Iodine reacts with starch to form a brownish-blue complex. Clear yellowish zones around the colonies indicate that the starch has been digested.
3. Examine the blood agar for hemolysis.

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4. Note the color of the agar and zones of cloudiness around the colonies on the MYP plates.
5. Note the pink coloration at the top of the uninoculated FTA tube: this coloration indicates the area in which the redox indicator, reazurin, is oxidized. Examine the growth patterns in the inoculated FTA tubes. Growth only at the top of the tube indicates an obligate aerobe. Growth throughout the tube is indicative of a facultative anaerobe. Growth in the bottom only is characteristic of an obligate anaerobe.
6. Read the TSI slants and record the results.
7. Remove 1 ml of MRVP culture and put into a new tube. Add 12 drops of VP reagent and four drops of 40% KOH and mix well. The appearance of red color within 30-60 min is indicative of acetoin production. (Note: the methyl red test is not valid for *Bacillus* species and should not be performed).

### STEP 7: Read the Results of the *E. coli* Plates and Compare Your Results With Your Gram+ Strain’s Results.