MicroLab Midterm

What does a defined medium contain?

Highly purified compounds

Laboratory benches should only be disinfected after finishing laboratory work. (T/F)

False

What should be included in a label of a bacterial culture?

name, date, organism, medium/test

Hands must be washed before and after lab. (T/F)

True

Before leaving the lab, what is your responsibility?

• Disinfect the bench-top

• Remove sharpie writing on tubes before discarding

• Wash hands

• Complete the results section of lab notebook

For your safety in lab you should…

Not eat or drink in the lab

What should be included in a label of a bacterial culture?

• Name or initials

• Date

• Name of organism

• Name of medium or test

Loops can be left in the incinerator until needed. (T/F)

False

Autoclaving sterilizes materials because of…

High heat and pressure

A bacterial colony is composed of genetically identical cells. (T/F)

True

For a simple stain, the heat-fixed slide is flooded with…

Either safranin (2 min) or crystal violet (1 min)

Steps of a simple stain (in order):

• Place water on slide

• Transfer bacteria with sterile loop

• Air-dry

• Heat fix

• Flood with safranin (2 min)

• Rinse and blot dry

When making a smear from liquid broth, first place water on slide. (T/F)

False

Simple stains are used to…

Increase contrast of cells

Crystal violet is the counterstain. (T/F)

False (safranin is counterstain)

Which pipettor is used for 195 µL?

P200

Gram-positive vs gram-negative staining difference is due to…

Cell wall

Which of the following is true about streak plating?

You must sterilize your inoculating loop between sections

Steps of Gram stain (in order):

• Air-dry smear

• Heat fix

• Crystal violet (1 min), rinse

• Gram’s iodine (1 min), rinse

• Decolorize with ethanol

• Safranin (2 min), rinse

Streak plate technique can be used to quantify bacteria. (T/F)

False

Bacteria with mycolic acids are mostly gram-negative. (T/F)

False

Acid-fast bacteria:

• Have a waxy, hydrophobic cell wall

• Contain a large amount of mycolic acid

Organisms growing in high salt concentrations are…

Halophiles

Statistically valid CFU range?

30–300

Dilution from 10 µL into 990 µL?

1:100

Fridge-spoiling bacteria are…

Psychrophiles

Bacteria that grow at 55–65 °C are…

Thermophiles

Spread plate results can quantify bacteria. (T/F)

True

Function of endospores?

Enable organisms to endure extreme temperature

Pseudomonas aeruginosa (CF pathogen, needs O₂) is a…

Obligate aerobe

MR-VP broth allows for how many tests?

2

Sulfur-oxidizers can reduce nitrate anaerobically. (T/F)

True

Catalase is found in…

Aerobes

Spore-formers are most likely…

Gram-positive

An organism growing with or without O₂ is an obligate anaerobe. (T/F)

False (that’s facultative)

Endospore stain slides should not be heat-fixed. (T/F)

False

Phenol red tests ability to ferment sugars. (T/F)

True

Thioglycolate medium shows surface growth + turbidity. Organism is…

Facultative anaerobe

TSI is differential for gram-negative enteric pathogens. (T/F)

True

Oxidase test identifies bacteria that…

Use cytochrome C as terminal electron acceptor

Endospores form instantly in boiling water. (T/F)

False

After entering lab, how do you prepare bench and self?

Wash hands, wear lab coat, disinfect bench, wear gloves, protect electronics

Why wear lab coat in microbiology lab?

Protect skin from harmful exposure

If leaving lab temporarily, what steps do you take?

Wash hands, remove lab coat before leaving

How do you dispose of plates and tubes?

Plates → biohazard bin (front of lab); Tubes → back of lab; remove writing/labels first

Why is handwashing important?

Prevents contaminating lab or carrying microbes home

At what temperature should microbes be incubated? Why?

20–25 °C for environment; 37 °C for pathogens (human body temp)

Define incubation.

Maintaining microbial cultures under controlled conditions

Define contaminant.

Unwanted microbe introduced into culture

Difference between defined and undefined media?

Defined = known purified compounds; Undefined = complex ingredients, composition unknown

What is a colony?

Visible mass of cells from one cell via binary fission

Total magnification with 40X objective + 10X ocular?

400X

How do you clean the microscope?

Lens paper, wipe after each use (low → high objective)

Fine vs coarse focusing knobs?

Coarse = large stage moves, 4X/10X; Fine = small stage moves, precise focusing, high power

Where do you place and secure slide?

On stage, secured with stage clips

What is aseptic technique?

Practices to transfer microbes without contamination

What is sterilization?

Chemical/physical process eliminating all living organisms

Four common sterilization methods?

Heat, radiation/gases, filtration, autoclaving

How do you evaluate broth/slants for contamination?

Check growth patterns, unexpected colonies/colors

Why mix broths before use?

Even distribution of bacteria/nutrients

Define inoculation.

Introducing microorganisms into sterile media

Why add oil with 100X objective?

Reduce light refraction, clearer image

Total magnification with 100X objective + 10X ocular?

1000X

Purpose of simple stain?

Increase contrast to view cells

What decolorizer is used in simple stain?

None

How is Saccharomyces cerevisiae different from bacteria in this lab?

Cells are larger than bacteria

Which micropipette for 1 mL sample?

P1000

If P200 set to 050, what volume is pipetted?

50 µL

Maximum volume for P20?

20 µL

What are differential stains?

Use differences in cell properties to distinguish groups

If iodine step skipped, what color is S. aureus? Why?

Red (lost CV complex during wash)

If decolorizer skipped, what color is E. coli? Why?

Purple (CV not removed)

Gram-positive vs gram-negative structural difference?

G+ thick peptidoglycan + teichoic acids; G– thin peptidoglycan + outer membrane rich in lipids

When use wet mount instead of gram stain?

To view live organisms, motility, natural arrangement

When use simple stain instead of gram stain?

To observe cell shape, size, arrangement only

How are acid-fast cells different from others stainable by gram stain?

Contain mycolic acids, waxy wall → resist gram stain and decolorization

What is a pure culture?

Culture of only one species, free of contamination

Why sterilize loop before streaking?

Remove contaminants

Why sterilize loop between streak sections?

Dilutes bacteria, isolates colonies, prevents transfer of too many cells

Purpose of streak plate?

Obtain well-isolated colonies from mixed culture

Why invert plate during incubation?

Prevent condensation from dripping on agar and mixing colonies

How do serial dilutions differ from streak plate?

Serial = dilute sample in liquid before plating; Streak = mechanical dilution on plate

If 10⁻³ plate had 20 colonies and 10⁻⁷ had 60 colonies, how many CFU/mL in original?

6.0 × 10⁸ CFU/mL (using countable plate range)

How many tubes to make 10⁻⁵ dilution with 1:10 dilutions?

5 tubes

If pellet had 3,500 CFU/mL and placed into 100 mL saline, how many CFU/mL?

35 CFU/mL

If 10⁻⁴ plate had 110 colonies, what is CFU/mL?

1.1 × 10⁶ CFU/mL

Why do different temperatures produce different growth rates?

Enzymes/proteins work within optimal range; outside → denature or slow

Define psychrophile.

Grows at 0–20 °C

Define mesophile.

Grows at 20–45 °C

Define thermophile.

Grows at 45–90 °C

Why do salt concentrations change growth rates?

Osmotic pressure changes; only halophiles thrive in high salinity

Define halophile.

Microbe growing optimally in high salt