Microbiology Laboratory Techniques: Media, Sterilization, and Staining

Practice questions covering media preparation, sterilization methods, microscopy principles, aseptic techniques, and various bacterial staining procedures.

What is the primary difference between a complex medium and a defined medium?

A complex medium is composed of digests of chemically undefined substances like yeast and meat extracts, whereas a defined medium has a precise chemical composition that is known.

What are the characteristics of Nutrient Agar ($pH\,6.8$) and Trypticase Soy Agar ($pH\,7.3$)?

They are complex, general-purpose media used for the isolation and cultivation of a variety of microorganisms.

Which medium is used specifically for cultivating fungi or yeasts?

Sabouraud Dextrose Agar ($pH\,5.6$).

What is the solidifying agent used in solid media, and from where is it extracted?

Agar, which is extracted from $Rhodophyta$ (red algae).

At what temperature does agar solidify after being heated to $100\,^{\circ}\text{C}$?

Between $40-42\,^{\circ}\text{C}$.

What are 'deeps' in the context of laboratory culture media?

Test tubes containing solid medium allowed to solidify in an upright position, usually used for anaerobic growth.

What is a selective medium?

A nutrient medium designed to favor the growth of certain microbes while inhibiting undesirable competitors.

How does a differential medium differ from a selective medium?

A differential medium provides a visible indication of a physiological characteristic, such as carbohydrate fermentation or enzyme production.

Which medium is both selective and differential for salt-tolerant organisms and mannitol fermenters?

Mannitol salt agar ($MSA$).

What does Eosin methylene blue agar ($EMB$) select for and differentiate?

It selects for Gram-negative bacteria and differentiates lactose fermenters (which take up dye) from non-lactose fermenters.

What are the standard operating conditions for an autoclave to achieve sterilization?

Heating to $121\,^{\circ}\text{C}$ at $15\,\text{psi}$ pressure for $15\,\text{minutes}-1\,\text{hour}$.

How does dry heat sterilization kill microorganisms?

It kills by oxidation effects, such as direct flaming of inoculating loops.

What is the temperature and duration required for hot-air sterilization of glassware?

Heating in an oven at $170\,^{\circ}\text{C}$ for $2\,hours$.

What method is used to sterilize heat-sensitive liquids or gases?

Filtration, using a membrane filter (composed of cellulose acetate or cellulose) with pores too small for microorganisms to pass.

What gas is commonly used for cold-sterilization of heat-sensitive objects like plastic Petri dishes?

Ethylene oxide.

How does ultraviolet ($UV$) radiation lead to the death of exposed organisms?

It causes damage to the $DNA$ by forming thymine dimers.

What is the primary purpose of a streak plate?

To isolate and maintain a microbial culture by diluting cells to achieve isolated colonies, which represent a pure culture.

How is total magnification calculated in a compound microscope?

It is the product of the magnification of the ocular lens ($10\times$) and the magnification of the objective lens (e.g., $100\times$), resulting in $1000\times$.

What is the maximum resolution of a compound light microscope?

Approximately $0.2\,\mu\text{m}$.

Why is immersion oil used with the $100\times$ objective lens?

It has the same refractive index as the glass slide and lens, which reduces light refraction and increases resolution.

What is the difference between direct rays and diffracted rays in microscopy?

Direct rays pass through a specimen unaffected, while diffracted rays are bent after passing through a sample.

What is aseptic technique?

A procedure performed under sterile conditions to avoid contamination of microbial cultures or sterile surfaces.

Why must the neck of a glass culture tube be passed through a flame?

To prevent contaminants from the air from entering the tube when it is opened.

What are the primary steps in preparing a bacterial smear?

Applying a thin layer of cells to a slide, air-drying the smear, and then heat-fixing it to kill the cells and adhere them to the slide.

Name the four reagents used in the Gram stain process in order.

Crystal violet (primary stain), Iodine (mordant), Ethanol (decolorizer), and Safranin (counterstain).

What color do Gram-positive and Gram-negative cells appear after a successful Gram stain?

Gram-positive cells appear purple, and Gram-negative cells appear red.

What specific component of the Gram-positive cell wall helps retain the crystal violet-iodine complex?

The thick layer of peptidoglycan.

What are endospores, and name two genera that produce them.

Specialized dormant, highly durable structures produced by genera such as $Clostridium$ and $Bacillus$.

Which primary stain and method are used in the Schaeffer-Fulton endospore stain?

Malachite green is used as the primary stain and is forced into the endospores using heat.

What unique lipid component is found in the cell walls of $Mycobacterium$ species?

Mycolic acids, which are complex branched-chain hydroxy lipids.

What is the primary stain used in the Ziehl-Neelson acid-fast stain?

Carbolfuchsin (a mixture of basic fuchsin and phenol).

What color do acid-fast and non-acid-fast cells appear after staining?

Acid-fast cells appear magenta (red), and non-acid-fast cells appear blue.

Why is egg albumin used when making a smear for an acid-fast stain?

The surface lipids of $Mycobacterium$ cause cells to form tight aggregates; egg albumin's high protein content helps disperse these cells on the slide.