Microbiology lab practical: Smear Prep and Gram staining and Quantitation Lab

What could happen if you overheat or underheat the slide during the heat-fixing process?

Overheat:

• damage/ distort cells

Underheat:

• cells aren’t fixed to the slide and can be washed off

• They’re alive

What happens if you heat fix a slide before drying it?

Boil the water and bursts the cells

Why is it important to heat-fix a slide before applying to stains?

• Adhere the bacteria onto the slide

• Kills the bacteria without destroying the structural features and helps stain penetrate better

How can an improperly prepared smear affect your gram stain results?

Staining procedure would be inaffective because the bacteria could have broken structures, be washed away to not heat-fixing, or still be alive, etc.

Primary stain and function?

Crystal Violet

• Penetrates the peptidoglycan layer in the gram (±)

Mordant and function?

Iodine

• Helps retention of the crystal violet 

• Supports the retention in gram (+) bacteria

Decolorizer and function?

Ethanol

• removes the crystal violet in gram (-) bacteria

Secondary stain and function?

Safranin

• pink color and promotes visualization of gram (-) bacteria

What equation was used to determine the CFU/ mL in lab?

#of colonies x 1/ dilution = __ CFU/ mL

if your plate had a total dilution of 10^-7  had 57 colonies, what is the CFU/mL in the original sample?

57 × 1/ (10^-7)= 5.7 x 10^8

Calculate the CFU/mL of the original sample if you followed the same steps as in the quantitation lab for the following plate: 

Number of colonies = 268 

Plate = total dilution 10^-5 

2.68 × 10^7

What’s the difference b/w direct and indirect quantitation?

Direct

• Physically counting the # of colonies

Indirect

• A measure on measuring WITHOUT counting them

Although we did not plate the 10^-1 tube in our Quantitation lab, based on your results what would you expect your 10^-2 plate to look like if you had?

the plate would contain a tenth less than the 1st one 

What factors can lead to a slowdown in bacterial growth despite initial exponential growth?

Bacterial growth slows when

• nutrients are exhausted

• wastes build up

• environmental conditions become unfavorable