Lab 2: Ubiquity Sampling, Aseptic Technique, Streak for Isolation, Smear Prep, and Gram Stain

A set of Q&A flashcards covering aseptic technique, streaking, smear prep, staining, Gram stain interpretation, and lab practices from the lecture notes.

In microbiology, what does a 'colony' represent?

Visible growth created by multiple rounds of bacterial reproduction; each colony originates from a single cell (pure culture).

What characteristics are described during Environmental Sampling?

Appearance, color, colony count, and other notes used to describe microorganisms.

What does beta-hemolysis indicate on blood agar?

Complete lysis of red blood cells with a clear zone around the colony.

What does alpha-hemolysis indicate on blood agar?

Partial lysis of red blood cells with a greenish discoloration around the colony.

What does gamma-hemolysis indicate on blood agar?

No hemolysis; growth with no red blood cell lysis.

Define aseptic technique.

A method of working with microbial cultures that prevents contamination of the environment, personnel, and cultures.

What is the difference between disinfection and sterilization?

Disinfection reduces microbial numbers to safe levels; sterilization removes all microbes, including spores.

What does 'sterile' mean in lab practice?

Complete removal of all microbes, including spores.

List some practices used to maintain aseptic technique in the lab.

Work area disinfection; sterilized loops and needles; proper culture tube handling; flame sterilization; handwashing; proper disposal and labeling.

What is the purpose of work area disinfection in the lab?

To destroy vegetative cells and viruses before and after work (not a sterilization).

Why is work area disinfection not considered sterilization?

Because it generally does not destroy endospores.

What is the purpose of bacteriological loops and needles, and how are they sterilized?

To transfer culture material; sterilized by heating in the incinerator until they glow orange, then cooled before use.

Why should you not store a loop in the incinerator?

The loop becomes extremely hot and can melt the rubber holder.

What is a pure culture?

A culture containing only one species of bacteria.

What is a mixed culture?

A culture containing more than one type of organism.

What is the purpose of the streak plate technique?

To thin out a small inoculum over the plate surface to separate individual cells that form colonies; each colony is a clone.

What is the typical quadrant streak plate sequence used in labs?

Quadrants 1 through 4; the loop is flamed and cooled between quadrants.

What is the purpose of preparing streak plates in microbiology?

To obtain a pure culture for definitive identification, DNA sequencing, and antibiotic sensitivity testing.

What bacterial species are used as a mixed culture in this lab?

Serratia marcescens and Escherichia coli.

What are the pure culture examples mentioned for agar plates?

Bacillus cereus or Staphylococcus epidermidis.

What is the role of TSA plates in streak plate experiments?

Provide a solid growth medium for observing colony morphology and isolation.

What is the purpose of the Gram stain in microbiology?

A differential stain that differentiates bacteria by cell wall structure (Gram-positive purple, Gram-negative pink).

List the main reagents used in Gram staining in order.

Crystal violet, Gram’s iodine, decolorizer (alcohol), and safranin.

What is the role of Gram’s iodine in Gram staining?

A mordant that forms an insoluble crystal violet–iodine complex to trap the dye in the cell wall.

Which step in Gram staining is critical for accuracy?

Decolorization with alcohol; timing is crucial.

What color do Gram-positive bacteria appear after decolorization and counterstaining?

Purple (they retain the crystal violet–iodine complex).

What color do Gram-negative bacteria appear after Gram staining?

Pink/red due to the counterstain with safranin after decolorization.

Why is Gram staining considered a gold standard in clinical microbiology?

Because it provides rapid, essential information about cell wall type to aid identification and treatment.

What are common Gram stain mistakes that can lead to false results?

Smear too thick; over-decolorization; using an old culture (over 16–18 hours) leading to shriveled peptidoglycan.

How should you handle staining pads during Gram staining?

Pads are used to hold stain; replace pads when full; 5-gallon bucket for disposal; do not rinse trays; place pads fluffy side up for better absorption.

How should you carry and care for a microscope properly?

Carry with two hands (one under the base, one on the arm); set gently on the bench; don’t let the cord hang over the table edge; turn off light; wipe oil; wrap cord; have instructor check.

What constitutes a good smear prep for staining?

Thin, evenly distributed cells that adhere to the slide; air-dried and heat-fixed.

What is the arrangement for cocci in irregular clusters, as described in the morphology chart?

Staphylococci (Gram-positive).

How can you describe neisseria sicca in the morphology chart?

Diplococci (Gram-negative) grouped in pairs/singular/tetrads

How can you describe cornyebacterium pseudodiphtheriticum?

Rod shaped in a v formation (Gram-Positive)

How can you describe enterococcus faecalis in the morphology chart?

Cocci in pairs or chains (Gram-positive)

How can you describe E. Coli in the morphology chart?

Rod-shaped (Gram-negative) in single or short chains.

How can you describe bacillus cereus in morphology chart?

Rod-shaped (Gram-positive) in chains or alone.