Reviewer – FS 106 Lab Final Exam

Complex Media vs. Chemically-defined Media

Exact chemical components are not known in Complex Media, thus it is better used in culturing an
unknown organism (Plate Count Agar – Beef Extract and Peptone)

If you want to know the Carbon source of S. aureus, use chemically-defined media since you know the
contents of the medium. It is usually used in studying the metabolic and nutritional properties of
bacteria

Bacteria – Plate Count Agar

Yeast and Molds – Acidified Potato Dextrose Agar

Agar – primary medium in culturing microorganisms

Sterilization Conditions: 121 deg. Celsius, 15 minutes, 15 psi

What is the difference between steam and air?

Steam is just purely water vapor, whereas air is a mixture of different compounds. Air is from the
atmosphere.

What happens if autoclave has air in it? (60% steam, 40% air)
Air decreases the internal temperature inside the autoclave. Thus, it would take a longer time to reach
the desired temperature of 121 deg. Celsius.
Even if pure steam and that which has air, have the same internal temperature, the one with pure steam
would still be able to sterilize better.
Air is heavier than steam thus it would be the one in contact with the container or microorganism, thus
decreasing the penetrating effect of steam inside the autoclave.

How would you know if the autoclave still contains air?

Pag sumingaw na, ibig sabihin nun steam na ang laman ng autoclave.
When steam already goes out of the hole, it signifies that there is no more air inside. Water is heavier
than air, thus when it is already heated too, it pushes the air out, and completely fills the autoclave with
steam.

Exercise 9: Not included in the exam

Exercise 10: Isolation methods

Streak Plate, Pour Plate, and Spread Plate – All are indirect methods in enumerating microorganisms
since you are not counting actual cells, you are counting the colonies formed.

Pour and Spread plate are used for enumeration. Streak plate is used for isolation – uses the concept of
dilution

Flaming in between streaks, so as to get a new set of microorganism for the new streak
New streak must come from the previous streak
-
Steps in performing pour plate: Inoculum before agar (1.0 mL)
- Formation of subsurface and surface colonies

Steps in performing spread plate: (0.1 mL) because smaller surface area
- overcrowding or visible spreaders (on the surface) would be the result if 1.0 mL is used in spread
plate

In using spread plate, the characteristics of colonies would be better observed

In using pour plate, there would be higher sensitivity. (When you are expecting lower counts)
- Since pour plate is 1 mL, 1 cfu/1 mL
- In spread plate, 1 cfu/0.1 mL. thus 10 cfu/ 1 mL

Example:
Sir Kenneth has suspension of mold that of high concentration. What plating technique must he use?
Answer: Spread plate, since molds are strictly aerobic organisms

Sir Kenneth has a psychrophilic organism. What plating technique must he use?
Answer: Spread plate, since organism may die if pour plate is used

Sir Kenneth is analyzing processed food. What plating technique must he use?
Answer: Pour plate, since you are expecting lower counts for processed food. You would rather employ
a technique that has a higher sensitivity

Direct Microscopic Count: Not included in the exam

When to use Plate Count Agar or Direct Microscopic Count?

Since you want faster results, plate count agar is not advisable since you still have to wait for 1 to 2 days
before you are able to read the results

Direct Microscopic Count usually used in pasteurized milk, more sensitive. Very small amount of results.
But may include dead cells in the counting

Plate Count Agar is used since there is lower risk of overestimating, only accounts for live cells.

Experiment 11-12: Microbiological Examination of Water and Food

Indicator Organisms > Actual Pathogenic Organisms

Water: Total Coliform, Fecal Coliform, E. coli (Most specific)

Are all total coliforms E. coli? No

Are all E. coli part of total coliforms? Yes

Total coliforms – Gram-negative, facultative anaerobes (can do fermentation), lactose producing acid
and gas (Salmonella)

Positive results: Turbidity and bubble formation in the broth

Fecal coliforms - Thermotolerant microorganisms (Incubated in an elevated temperature)
E. coli – predominant facultative anaerobe in the human body, 99% from the body, specifically feces

LST -> BGLB broth -> EC broth

LST is used as resuscitation part for injured organisms. If directly placed in BGLB, result may indicate a
false negative.

Experiment 15: Control Methods

Antiseptic: Used on living organisms

Disinfectant: Used on inanimate objects

Mechanisms of Action – interferes with proteins

Iodine – hinders the production of protein, instead of amino acids binding to each other, iodine is the
one that forms complex with the amino acids, inhibiting the formation of polypeptide

Soap – formation of micelles. Mechanical removal of the organisms, does not really kill the
microorganisms. Solubilizes oil, and is able to mix with the compounds
- percent microbial growth reduction (by micelle formation)
- hydrophilic head and hydrophobic tail, surrounds the dirt, goes with the rinsing

Alcohol – functions by Hydrogen bond disruption

- Hydrogen bonding determines the folding of the protein, when H-bonds are destroyed, the
proteins are unfolded, thus destroying the structure

Domex – NaOCl
- denatures protein through oxidation, Chlorine is an intense oxidizing agent

Penicillin – affects the formation of cell wall (peptidoglycan)

- Peptidoglycan is composed of alternative layers of NAM and NAG, binding proteins.
- Binding proteins now attach to the penicillin, destroys the integrity of the cell wall

Sodium Nitrite
- inhibits clostridium botulinum endospores

Acetic Acid
- change of pH, precipitating the proteins whilst destroying the bonds of the nucleic acids,
destroying the manufacturing of proteins

Experiment 15 - Parasitology –
*study the life cycle of the organisms for the exam

Definitive host – where organisms grow, where microorganism matures, and where parasite generally
resides
Intermediate host – used to go to the next stage for the microorganism
Protozoans used in study: (one-celled)
Entamoeba histolytica
Giardia lamblia
- multiply through trophozites

Helminths (multicellular)
Schistosoma
- produce eggs, but hatches only when exposed to the environment
- perhaps through repeated exposure to the environment that the helminth is able to thrive in
the body