Lab Assignment 3
Lab Assignment 3
Lab Assignment 3
Answer the ques,ons directly in the space provided. Do not a8ach addi,onal pages.
By entering your name below, you cer,fy that you have completed the lab yourself, and the answers
given are your own. Copying assignments of other students, or other forms of plagiarism will result in a
grade of zero, and will be reported to the Faculty of Science.
1. What is the easiest way to put just the right amount of water on a microscope slide when you’re
making a smear from bacteria grown on a Petri plate? (0.5 marks)
Turn the tap un,l it's slowly dripping and catch a loop full with the loop smear s,ck to place onto the
microscope slide.
2. Why should you always make your smear from a well separated colony, rather than from a place on
the plate where colonies are growing together? (1 mark)
You always make your smear from a well separate colony to exercise restraint. If it was taken from a
place on the plate where colonies have grown together, you will pick up way too many cells. A thick
smear will prevent the stains from penetra,ng through all the cells.
3. Give two differences between the cell walls of Gram posi,ve bacteria and Gram nega,ve bacteria. (1
mark)
i. Gram posi,ve bacteria have a thick layer of pep,doglycan, while Gram nega,ve bacteria have a
thin layer of pep,doglycan.
ii. Gram nega,ve bacteria have a semi-permeable outer membrane, while Gram posi,ve bacteria
do not.
4. What is the mordant in the Gram stain? What does the mordant do? (1 mark)
The mordant in the gram stain is iodine. The mordant acts as a helper to increase the intensity of the
primary stain. Iodine acts by forming complexes with the crystal violet par,cles, causing them to
increase in size and be trapped more effec,vely by the pep,doglycan.
5. If you skipped the differen,al step in your Gram stain, and went directly from step 2 (iodine
treatment) to step 4 (counter staining), what color would you expect the following types of cells to
be? (1 mark)
6. Examine the Gram stain of Staphylococcus epidermidis shown in the online lab. What is the Gram
reac,on, cell shape and arrangement? (1.5 marks)
Gram reac,on: __purple stain (Gram posi,ve)____Cell shape: ___cocci _______
Arrangement: ___staphylococci____
7. Why should you do your Gram stain with an overnight culture whenever possible? What is the risk of
using older cultures? (1 mark)
Staining an overnight culture ensures that the bacteria culture is fresh and from a single colony. If old
cultures are used, there is a large chance of there being unhealthy or dead bacteria which stain a
different colour and are prone to giving incorrect results.
8. What number is your Pet bacterium? Describe the Gram reac,on, cell shape and arrangement that
you observed in the Gram stain of your Pet bacterium. (2 marks)
Unknown number: 30
Gram reac,on, cell shape and arrangement (if applicable): Purple stain (Gram posi,ve), bacilli, pairs
(diplobacilli)
9. What molecule is responsible for the strength and rigidity of the bacterial cell wall? (0.5 marks)
Pep,doglycan
10. What kind of cells are more likely to be broken open by a base in the KOH string test? (0.5 marks)
The gram nega,ve cells, with their thinner pep,doglycan cell wall.
11. What was the result of the KOH test for your Pet bacterium? Stringy or not stringy? (0.5 marks)
☐ Stringy ☐ Not stringy
12. What does this tell you? (0.5 marks) Bacteria is Gram posi,ve
13. Why did the bacteria need to be resuspended in a tube of T-soy broth, and then placed in the
incubator about 45 minutes before the hanging drop slides were made? (1 mark)
To give cells enough ,me to return to the logarithmic phase of growth, giving us an “early log-phase
culture” that should allow us to see bacterial mobility under the microscope.
14. You prepare a hanging drop slide from an unknown bacterium. When you look at it with your
microscope, you see that the cells are shaking and they’re all moving in the same direc,on. Are they
mo,le? Explain your answer. (1.5 marks)
The unknown bacterium is not mo,le. The shaking is a result of Brownian mo,on; the random
movement of par,cles in liquid. As well, if they’re all moving in the same direc,on this is a result of
current in the hanging slide, not their own movement pa8erns.
15. What is the func,on of the dye 2,3,5-triphenyltetrazolium chloride in the mo,lity agar stab tube?
What does the dye show us? (1 mark)
The dye acts as an electron acceptor, and turns red in the presence of metabolically ac,ve bacteria.
It will show us where the bacteria are, and where they’ve gone inside the tube.
16. Describe the results of the mo,lity tests for each of the following three bacteria: (3 marks)
Your pet bacterium Bacteria is mo,le. The running and Bacteria is mo,le. The red colour has
tumbling mo,on of bacterial mo,lity spread away from the stab line.
is present.
# ____30_______
17. Give the flagellar arrangement for each of the following bacteria: (1 mark)
18. Examine the picture showing a capsule stain of Klebsiella pneumoniae. Describe the appearance of
the cell, the capsule and background of the slide. (1.5 marks)
The cells have a bacillus shape and most appear to be successfully stained violet by the crystal violet
stain. The capsule is visible as a colourless barrier around the cell. We can see the capsule because of
the nigrosin stain that appears greyish in the background of the cells.
19. How would the capsule stain look different if you omi8ed the crystal violet? Would you s,ll be able
to see the capsule? Explain. (1.5 marks)
The crystal violet stain is used to penetrate the capsule and stain the cell. If we omi8ed the crystal violet
stain, the en,re bacteria would appear colourless against the background of the slide. Therefore the
capsule would not be dis,nguishable.
20. Describe the expected colour of the cells, the endospores, and the background aler performing the
following staining procedures on a 48-hour old culture of B. sub;lis: (3 marks)
Specimen Classic spore stain Simple spore stain Simple stain with safranin
(with malachite green) (with permanent marker) (like in Exercise 6)
Mother cells Pink Pink Pink
21. How do you think the results of the spore stains would have differed if we used a 16-hour old culture
of B. sub;lis? (Instead of the 48-hour old culture.) Explain why. (1 mark)
Endospore forma,on is usually triggered by the lack of nutrients when growing cells run out of food. If
we used 16-hour old culture instead of 48-hour old culture the environmental condi,ons may s,ll be
op,mal, therefore the cells won’t begin the process of endosporula,on. Meaning we would see very
small to no endospore produc,on.
Total = / 27