lab practical review

Question 1

gram stain procedure

Answer 1

1. crystal violet penetrates cell wall and plasma membrane of both types of cells
2. Gram’s iodine binds to crystal violet, forming an insoluble precipitate that locks purple color into cells
3. ethyl alcohol dehydrates the peptidoglycan layer and dissolves lipid-containing layers
   * *peptidoglycan layer shrinks and traps violet in Gram +
   * *peptidoglycan layer too thin, dye washes out of Gram -
4. safranin turns Gram - pink, while violet in Gram + masks the dye and they stay violet

Question 2

gram + vs gram - cells and examples

Answer 2

gram +: thick peptidoglycan layer (M. luteus)

gram -: thin peptidoglycan layer, more complex cell wall with a lipopolysaccharide layer (E. coli)

Question 3

antibiotic types, how they work, and which bacteria they work on

Answer 3

penicillin

• beta lactam antibiotic
• works on Gram + (M. luteus)
• inhibits transpeptidase enzyme which links chains in the peptidoglycan layer (death by osmosis)
• not effective on Gram - because peptidoglycan layer is smaller part of cell wall

streptomyosin

• aminoglycoside antibiotic
• works on Gram - (E. coli)
• changes ribosome structure, causing mRNA to be read incorrectly (producing nonfunctional proteins)
• not effective on Gram + because it’s too large to get through the peptidoglycan layer

Question 4

running ANOVA

Answer 4

• “treatment” in 1st column, number them (1, 2, 3, etc)
• dependent variable “response” in 2nd column corresponding to treatment
• stat–anova–general linear model–fit general linear model
• dependent variable in “response” box, independent variable in “factors”
• gives F and P-value, do Tukey if p < 0.05

Question 5

running Tukey

Answer 5

• stat–anova–general linear model–comparisons
• ensure: pairwise comparison, “treatment” is checked for terms, Tukey checked
• options–should be 95% confidence level
• graphs–interval plot for differences should be unchecked
• results–uncheck grouping info, check tests and confidence intervals

run test!

Question 6

parts of a conclusion statement

Answer 6

• what did you expect to find (hypothesis)
• what did you find? (f-value, p-values, figure reference) - did it match expectations?
• can you accept hypothesis? (use words “support” or “fail to support”)
• if accepted–broader scientific applications; if not accepted–biological explanations for unexpected findings

Question 7

hemacytometer use

Answer 7

focusing hemacytometer (before sample added)

• raise stage as high as it will go, and move down to focus
• triple line border distinguishes large corner squares

use

• remove slide after focusing microscope and put cover slip on
• use special “cut” pipette tip to load mixture into hemacytometer by placing the tip at the edge of these coverslip (works by capillary action)

Question 8

calculating dilution factor and cell concentrations in hemacytometer

Answer 8

dilution factor
DF = v2 (total after) / v1 (volume of concentrated stock solution)

*area of the major corner squares is 0.0001 ml

cell concentration = (average # of cells)(DF)(1 x 10^4) cells/ml stock solution