Microbiology: Lab Review Chapter 4,5,6

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Chapter 4 Laboratory Review Questions

1.    [CH-04-01] Why is it important to make the bacterial smear thin? – Making bacterial smear thin prevents the overcrowding of the slide and allows seeing the individual bacterial cells and their growth arrangements.

2.    [CH-04-02] What is the purpose of heat-fixing the smear? – Heat-fixing kills bacterial cells and makes them stick to the slide.

3.    [CH-04-03] Why is it important to dry the smear before heat fixing? – if smear is wet during heat-fixation, it will overheat and boil causing rupture of bacterial cells. The bacterial cells would also be washed away during staining procedure as they will not stick to the slide.

4.    [CH-04-04] How do methylene blue and other basic dyes attach to the bacterial cell? – methylene blue and other basic dyes are positively charged which allows them to bind to negatively charged surface of bacterial cell.

5.    [CH-04-05] Do you think a dye with a negative charge could be used in simple stain of bacteria? Explain your answer – No. Surface of bacterial cell is binding the dye in simple stain. It is negatively charged. Therefore, it will repel negatively charged dye.  

6.    [CH-04-06] Distinguish between cell morphology and cell grouping – Cell morphology is describing the cell shape that can be in form of cocci, bacilli. Cell grouping is describing the cell arrangements with respect to each other that can be in form of pairs, chains, clusters, packets, tetrads

7.    [CH-04-07] Describe morphology, cell grouping, oxygen and temperature requirements for Bacillus subtilis – Bacilli without growth arrangement, mesophilic obligate aerobe.

8.    [CH-04-08] Describe morphology, cell grouping, oxygen and temperature requirements for Escherichia coli – bacillus with no cell grouping, that is mesophilic facultative anaerobe.

9.    [CH-04-09] Consider a coccus and a bacillus of equal volume. Which is more likely to survive in a dry environment? Explain your answer – Cocci are better adapted to survive in dry environment as they have lower cellular surface to volume ratio enabling them to reduce the water loss

10. [CH-04-10] Consider a coccus and a bacillus of equal volume. Which one is better adapted to a moist environment? Explain your answer – Bacillus is better adapted to moist environment as it has higher cellular surface to volume ratio enabling them more efficiently absorb nutrients.

Chapter 5 Laboratory Review Questions

1.    [CH-05-01] Why is Gram staining a differential stain? – The stain divides bacteria into separate categories based on the structure of their cell wall. Allows for the basic differentiation of bacteria based on type of cell wall.

2.    [CH-05-02] Which of the reagents used in the Gram stain is the:

1.       counter stain            safranin
2.       decolorizer                95% alcohol
3.       mordant                     Gram’s iodine
4.       primary stain            crystal violet

3.    [CH-05-03] Explain the term mordant – A chemical compound that forms a complex with a dye, thus retaining it in the object.

4.    [CH-05-04] Compare the cell wall of Gram-positive and Gram-negative bacteria – Gram-positive bacteria have a thick peptidoglycan layer with teichoic acid on a single cytoplasmic membrane. Gram-negative bacteria have thin peptidoglycan layer between an outer membrane and an inner cytoplasmic membrane and outer membrane has a layer of lipopolysaccharides on top.

5.    [CH-05-05] In Gram stain, why does alcohol decolorize Gram-negative bacteria? –Gram-negative bacteria have an outer membrane that breaks down in 95% alcohol releasing the crystal violet. In gram-positive bacteria the thick peptidoglycan layer protects and that better prevents the breakdown of the membrane and leaking of the crystal violet.

6.    [CH-05-06] In Gram stain, if you stopped the procedure after the crystal violet is applied but prior to decolorization how would the Gram-negative bacteria appear? How would the Gram-positive bacteria appear? –

The gram-negative and gram-positive bacteria would both appear violet as the alcohol wash was not applied to remove the dye.

7.    [CH-05-07] In Gram stain, if you stopped the procedure after decolorization how would the Gram-negative bacteria appear? How would the Gram-positive bacteria appear? –

Gram-positive bacteria would appear violet as their thick peptidoglycan layer enables them keep the crystal violet dye bonded to the membranes better. Gram-negative bacteria would appear clear and transparent, as the decolorization has cleared the crystal violet dye out of them.

8.    [CH-05-08] In Gram stain, if at the end of the procedure both the Gram-positive and Gram-negative bacteria appear red what is the most likely explanation? –

Too much of the decolorizer was added to the sample, washing away the crystal violet from the gram-positive bacteria. Safranin that was added later ended up coloring both gram-positive and gram-negative bacteria red.

9.    [CH-05-09] Name clinically important (pathogenic) Gram-positive bacterial species –

Streptococcus pyogenes

Staphylococcus aureus

Bacillus anthracis

Clostridium botulinum

Clostridium tetani

Clostridium difficile

Corynebacterium diphtheriae

Listeria monocytogenes

10. [CH-05-10] Name clinically important (pathogenic) Gram-negative bacterial species –

Neisseria meningitides

Neisseria gonorrhoeae

Borrelia burgorferi

Vibrio cholerae

Legionella pneumophilia

Escherichia coli

Heliobacter pylori

Salmonella enteritidis

11. [CH-05-11] Explain the terms false Gram-positive and false Gram-negative. When can you get false Gram-positive stain? –

The false addition is exactly what it sounds like. Getting a false gram-positive/negative result. Possible to get a false gram-positive stain by forgetting to add 95% alcohol to wash the specimen or forgetting to counter stain with Safranin.

12. [CH-05-12] Name two reasons of getting false Gram-negative stain results –

- Adding too much decolorizer

- Forgetting to add the mordant

13. [CH-05-13] If you performed the Gram stain on human epithelial cells what color do you think would result? –

Pink because the human cells do not have cell walls to retain the dyes and would end up with the last dye on the membrane.

14. [CH-05-14] Both crystal violet and safranin are basic stains and can be used in simple stain of Gram-positive and Gram-negative bacteria. Explain how they end up staining Gram-positive and Gram-negative cells differently in the Gram stain –

In a Gram stain, there is either the assumption that the sample will contain both gram-positive and gram-negative bacteria or the sample is composed of an unknown. Thus, the sample is treated with both the crystal violet and the safranin to determine which dye ends up staying in the bacteria.

Chapter 6 Laboratory Review Questions

1.    [CH-06-01] What is the difference between positive and negative staining? – Positive staining, targets the membranes of bacteria cells to highlight the bacteria. Negative staining, uses the repulsion of charges to target the background color of the image leaving the cell in sharp contrast to the background.

2.    [CH-06-02] Distinguish between cell membrane, cell wall, and capsule –

Cell membrane – semipermeable membrane all cells have to protect their inside cytoplasm from the environment.

Cell wall – Additional cell structure surrounding the outside of the cell membrane. Present in plants, bacteria and archaea.

Capsule – An additional polysaccharide layer that lies outside the cell envelope that helps bacteria avoid be phagocytosis.

3.    [CH-06-03] Capsulated Streptococcus pneumoniae cause pneumonia in mice whereas non-capsulated Streptococcus pneumoniae are harmless. Explain this phenomenon –

Capsuled version of Steptococcus pneumoniae has an increased virulence factor due to its capsule. The capsule helps protect the bacteria against the immune system’s macrophages. Therefore the capsulated form is more capable of causing disease.

4.    [CH-06-04] Give examples of vaccines that consist of capsular substance –
Pneumococcal polysaccharide vaccine (PPSV) against Streptococcus pneumoniae
Pneumococcal conjugated vaccine against 7 or 13 antigens against S. pneumoniae
Meningococcal polysaccharide vaccine against 4 strains (MPSV-4) of Neisseria meningitidis

5.    [CH-06-05] What are the functions of capsule in bacterial cell? –

- Help cells avoid phagocytosis by macrophages

- Contains water to prevent desiccation

- Exclude bacterial viruses, and hydrophobic toxins

- Help bacteria adhere to surfaces

6.    [CH-06-06] Why does not a negative stain colorize the cells in the smear? –

The negative stain does not colorize the cells in the smear because of repulsion of like charges.