COLIFORMS, STAPH, AND STREP

EXAMINATION OF WATER SAMPLES FOR COLIFORMS

This is a procedure to determine the presence of fecal pollution in water. It is used to look for coliforms, and it is a fecal indicator for bacteria.

Characteristics of coliforms

  1. Enteric (found in the intestines)
  2. High numbers
  3. easy to ID
  4. Gram negative rods, non-spore forming
  5. Ferment lactose
  6. Produce acid and gas products
  1. Presumptive Test: Phenol Red Lactose Broth
  2. Inoculate the broth with a water sample.
  3. The results are qualitative (determines if there are coliforms present)
  1. Most Probable Number Determination
  2. The results are quantitative (determines how many coliforms are present)

Procedure

Inoculate 9 test tubes with water sample

1.1000 μl of water sample to each of three tubes of PRL broth

2.100 μl of watersample to each of three tubes of PRL broth

3.10 μl of water sample to each of three tubes of PRL broth

Two lab tables will use only water sample A for their tubes.

Two lab tables will use only water sample B for their tubes.

Two lab tables will use only water sample C for their tubes.

Put all 9 tubes from your lab group in one rack, place in incubator.

Expected Results:

Phenol Red will turn yellow in the presence of acid, which indicates fermentation of lactose.

Each PRL tube also contains a small, inverted tube called a Durham tube inside of it. If gas is produced, a gas bubble will appear within the Durham tube by the next lab period. If the lactose is fermented, the broth will turn yellow as well.

A positive test (yellow and gas) is expected from coliforms. (However, there are some Gram positive organisms that can be positive as well. That’s why we usually plate out the water sample on an EMB plate, to inhibit the growth of Gram positive bacteria.)

SELECTIVE AND DIFFERENTIAL MEDIA

A media that is selective is one that inhibits other bacteria so only the types you want can grow.A media that is differential contains ingredients (chemical indicators) that produce observable differences between species of bacteria that are present.

Examples of media which are both selective and differential

  1. EMB and MacConkey’s Agar
  2. MSA

Example of media which is only differential

  1. BLOOD AGAR

EOSIN-METHYLENE BLUE AGAR (EMB)

This medium is used to isolate Gram negative coliforms.

It contains eosin and methylene blue (which are stains) and lactose (a sugar). If the lactose is fermented, the acid products will cause the stains to go from colorless to pink.

Selective: stains allow only Gram negative to grow.

Differential: when lactose is fermented, the phenol red causes a color change.

Lactose Fermentation (pink color change)

E. coli (will also cause the stains to have a green sheen)

Enterobacter (pink colonies will have a central purple spot (fish-eye colonies)

No Lactose Fermentation (colorless)

Proteus vulgaris and Salmonella

MANNITOL SALT AGAR (MSA)

This medium is used to isolate Staphylococcus.

It contains salt, mannitol (a sugar), and phenol red.

Selective: salt allows only Gram positive staphylococcus to grow.

Differential: when mannitol is fermented the phenol red causes a color change.

Mannitol Fermentation (color becomes yellow)

Staphylococcus aureus

No Mannitol Fermentation (color stays red)

Staphylococcus epidermidis and Micrococcus

BLOOD AGAR

This medium is not selective; it is differential for hemolysis, useful in identifying Streptococcus.

It contains sheep blood.

Differential: according to type of hemolysis (destruction of red blood cells)

Alpha hemolysis: green halo around colonies (partial breakdown of RBCs)

Example: E. coli

Beta hemolysis: clear zone around colonies (complete breakdown of RBCs)

Example: Streptococcus pyogenes (“strep throat”)

Gamma hemolysis: no hemolysis

Example: Staphylococcus epidermidis

CULTURE OF STAPHYLOCOCCUS

Each of you will get two sterile cotton swabs in a packet. Use the first swab to take a sample from inside your own nose (don’t touch the outside skin). The second swab will be used for the next procedure on the throat.

Procedure

  1. Label the MSA plate (grease pencil) with your name and lab time. Mark the quadrants.
  2. Label a staph broth tube with tape (leave a handle) with your name, “nose”, and lab time.
  3. Obtain the nasal sample on a cotton swab.
  4. Roll the swab over the first quadrant.
  5. Place the swab inside a test tube of staph broth.
  6. Break the stick off so it will fit in the tube.
  7. Place the cap on the tube.
  8. Place in rack and incubate.
  9. Flame a loop, cool in the side of the agar, and re-streak over the first quadrant.
  10. Flame again, cool in the side of the agar, and streak the second quadrant.
  11. Flame again, cool in the side of the agar, and streak the third quadrant.
  12. Flame again, cool in the side of the agar, and streak the fourth quadrant.

CULTURE OF STREPTOCOCCUS

Procedure

  1. Label the Blood Agar plate (grease pencil) with your name and lab time. Mark the quadrants.
  2. Label a strep broth tube with tape (leave a handle) with your name, “throat”, and lab time.
  3. Use the wooden tongue depressor on your lab partner to hold their tongue down.
  4. Tell them to look up toward a light, stick out their tongue, and say “Ah”.
  5. Use the sterile cotton swab to gently obtain a sample from the palatine tonsil area, just lateral to the uvula.
  6. Roll the swab over the first quadrant of the plate.
  7. Place the swab inside a test tube of strep broth.
  8. Break the stick off so it will fit in the tube.
  9. Place the cap on the tube.
  10. Place in rack and incubate.
  11. Flame a loop, cool in the side of the agar, and re-streak over the first quadrant.
  12. Flame again, cool in the side of the agar, and streak the second quadrant.
  13. Flame again, cool in the side of the agar, and streak the third quadrant.
  14. Flame again, cool in the side of the agar, and streak the fourth quadrant.
  15. Tape both plates together (leave a tape handle) and incubate.

EVALUATION OF WATER, ALCOHOL, STAPH, AND STREP

Fill out your handouts today for these experiments

Handout Report 48: Bacteriological Examination of Water (For Coliforms)

The goal is to determine if water has fecal pollution by looking for coliforms, which are a fecal indicator of bacteria. Remember, coliforms are Gram negative, non-spore forming rods. The ferment lactose and produce gas and acid. To declare the presence of coliforms, we need to run a series of three tests.

1A) Presumptive Test: this checks for fermentation and gas by inoculating phenol red lactose broth with the water sample. Phenol Red is a pH indicator to show the presence of acid. Lactose is needed as a substrate for evidence of fermentation. A Durham Tube is also needed to check for gas production.

1B) Quantitative Test: Most Probable Number (MPN). Inoculate 9 different tubes with three different columes: 1000μl (1ml), 100μl (0.1 ml), 10μl (0.01ml). Check to see how many of your tubes are positive (yellow and gas). Record them in this table:

1ml / 0.1ml / 0.01ml
Sample A
Sample B
Sample C

To calculate MPN, look at the table in your handout. For example, if sample A had a result in the table above of 3-3-0, then see on the handout table that it corresponds to 240 colonies in 100ml of sample. However, we used a volume that was 1/10th of that, so our dilution factor is 0.1. Therefore, 240/0.1 = 2400 coliform colonies per 100ml.

2) Confirmed Test: Inoculate a selective and differential medium to eliminate false positive results. False positive results can occur from Gram positive organisms that ferment lactose and gas, but since they are not coliforms, we do not want to count them. Therefore, we must inoculate a medium that inhibits Gram positive organisms and allows differentiation of lactose fermentors. For this, we use Levine’s EMB Agar. It contains two selective agents to inhibit Gram positive organisms: eosin and methylene blue. It is also differential because lactose fermenters show up as blue-black colonies when you look at them through transmitted light (hold the plate up to the light and look at them). The acid causes them to be black in the center of each colony = nucleated (fisheye) colony. This is indicative of a positive test. Examples of coliforms that will be nucleated are E. coli, Citrobacter, and Enterobacter.

One organism we have studied (E.coli) produces a great deal of acid. Remember that E. coli is Methyl Red positive, and MR only turns red at very acidic pH (less than 4.5). In excess acid, it will show a green metallic sheen. This is also positive for a confirmed test.

3) Completed Test: Take a positive colony from the EMB agar and inoculate two media:

NA Slant: Do a Gram stain. Gram negative rods are a positive test.

PR Lactose broth: Acid and gas production after 24 hours is a positive test.

Both of these tests must be positive for a positive completed test.

Handout Lab Report 55: Staphylococci

Why did we use a nasal sample? Because 20% of the human population normally carries Staphylococcus aureus in their nose. The media we used was MSA. It contains salt (selective) which inhibits everything but Staphylococcus (S. aureus, S. epidermidis, and Micrococcus). It also contains mannitol (differential) because only Staph aureus and Staph epidermidis ferment mannitol (makes the media yellow).

Therefore, if the MSA plate tuned yellow, we still need to discern between S. epidermidis (non-pathogenic) and S. aureus (pathogenic). For this, we do a coagulase test. This test is a tube of rabbit plasma. A loop of a colony from the yellow area of the plate is added, and the tube is incubated for 6 hours. When you tilt the tube and the plasma is solidified and does not run, it is positive for coagulase.

Coagulase negative indicates Staphylococcus epidermidis.

Coagulase positive indicates Staphylococcus aureus.

Handout Lab Report 56: Streptococci

Check your plates under the colony counter for signs of alpha hemolysis (greenish clearing areas), beta hemolysis (clear areas), or gamma hemolysis (no clearing).

Beta Hemolytic streptococci / Bacitracin sensitivity / CAMP test
Group A (Strep pyogenes / + / -
Group B (Strep agalactiae) / - / +
Alpha Hemolytic streptococci / Optochin sensitivity / Bile Solubility test
Strep pneumoniae (virulent) / + / +
Strep viridans (non-virulent) / - / -

Strep pyogenes causes Strep Throat.

Strep agalactiae usually only causes disease in newborns. It can be transmitted vaginally from the normal biota of the mother, and transmitted in milk.

Strep pneumoniae causes pneumonia.

Strep viridans does not cause disease and is normal mocrobiota.

CAMPTEST

This test differentiates between Groups A and B Streptococcus. Use a Blood agar plate, and streak a known sample of Staph aureus in the lower 1/3 of the plate. Then make a perpendicular streak of the suspected Group B that almost touches the previous streak. Incubate. Staph aureus is strongly beta hemolytic because it has hemolysins. Streptococcus is weakly beta hemolytic from an exotoxin called streptolysin. When these two hemolysins come in comtact with each other, they enhance each other’s beta hemolysis, and the clearing forms an arrow. This is a positive CAMP test.

ANTIBIOTIC SENSITIVITY TESTS (Bacitracin and Optochin)

A disc with the antibiotic in it is placed on the plate where the colonies are. If there is a zone of clearing, it indicates sensitivity to the antibiotic, and can be killed by it.

BILE SOLUBILITY TEST

Bile is added to a colony. If the colony dissolves, the test is positive.

Handout Lab Report 32: Alcohol Evaluation

To evaluate the effectiveness of alcohol’s antimicrobial activity, we need to calculate the percent reduction.

(colony count of first press) – (colony count of second press)

% Reduction = Colony count of first pressx 100

Example

First press of left thumb = 32 colonies

Second press of left thumb = 7 colonies

(32-7) / 32 x 100 = 78%

Therefore, pressing your left thumb removed 78% of the colonies. This will serve as a control.

The right thumb is the experimental side.

First press of right thumb = 13 colonies

Second press of right thumb = 0 colonies

(13-0) / 13 x 100 = 0%

That means that the alcohol removed 100% of the bacteria. Compared to 78%, alcohol was 22% more effective.

Mode of Action of Alcohol:

  1. Alcohol dissolves the oil (sebaceous oil) in the skin, and the bacteria are physically rubbed off with it.
  2. Alcohol disrupts the plasma membrane of the bacteria, which is composed of phospholipids.

Handout Lab Report 33: Antimicrobic Sensitivity Testing (Kirby-Bauer)

Your plate was heavly seeded with one of the following organisms:

Staph aureus

Pseudomonas auriginosa

Proteus vulgaris

E. coli

Then you put antibiotic discs on with the following codes:

C-30 / Chloramphenicol
K-30 / Kanamycin
GM-10 / Gentamycin
E-15 / Erythromycin
P-10 / Penicillin
Te-30 / Tetracycline
AM-10 / Ampicillin
S-10 / Streptomycin

Check and see how many clear zones (zones of inhibition) you have from your discs. Measure each zone (the complete diameter) with a ruler in mm. If you do not have a full diameter because your disc is too close to the edge of the plate or to another disc, measure the radius and multiply by two. Compare your measurements to the table in the handout (VIII) and write down on your report whether the organism was resistant, intermediate, or sensitive to the antibiotic disc.