Microbiology Lab Review: Chapters 7,8

Lab review

Chapter 7 - Endospore stain

1. [CH-07-01] For acid-fast stain, name the
1. Primary stain                        Carbol Fuchsine
2. Decolorizer                           Acid alcohol
3. Counterstain                        Methylene blue

2.    [CH-07-02] How does the decolorizer in the acid-fast procedure differ from the decolorizer in the gram stain? –

            Instead of 95% alcohol, the decolorizer in this situation is acid alcohol, a combination of alcohol and acid. The acid alcohol is needed when dealing with acid-fast bacteria because like dissolves like.

3.    [CH-07-03] If at the end of the procedure all the bacteria (known to be a mixture of acid-fast and non-acid-fast bacteria) stain red, what technical mistake was made? –

The specimen was allowed to dry out during the steaming process resulting in carbol fuchsine being stuck onto all bacteria in the sample.

4.    [CH-07-04] Describe the morphology, cell grouping, oxygen and temperature requirements for Mycobacterium smegmatis –

            Bacillus, Acid-fast bacterium, obligate aerobe

5.    [CH-07-05] Name the pathogenic species of acid-fast bacteria and the disease each species causes –

            Mycobacterium africanum à TB

            Mycobacterium bovis à TB

            Mycobacterium tuberculosis à TB

            Mycobacterium marinum à skin infection

            Mycobacterium avium à atypical mycobacterial infections

            Mycobacterium kansasii à chronic human pulmonary diseases

6.    [CH-07-06] What substances in the wall of acid-fast bacteria account for their staining reaction? –

            Mycolic acid and multiple lipopolysaccharide layers account for acid-fast bacteria’s unique staining reaction, which requires heat for the dyes to get into the cell.

7.    [CH-07-07] Why are mycobacteria more resistant to disinfectants than other vegetative bacteria? –   

            Their bacterial cell walls are composed of thick layer of peptidoglycan along with lipopolysaccharide layer, layer of hydrophobic mycolic acids, and a capsule. All these components make the mycobacteria’s cell envelope highly hydrophobic and prevents disinfectant solutes from passing into the cell.

8.    [CH-07-08] Compare the cell wall of acid-fast bacteria with the cell wall of Gram-positive and Gram-negative bacteria –

Acid fast: Cell membrane + thick peptidoglycan + Lipopolysaccharide + mycolic acid & long lipids + capsule + Lipopolysaccharide

Gram positive: Cell membrane + Thick peptidoglycan + teichoic acid

Gram negative: Inner cell membrane + Thin peptidoglycan + outer membrane + Lipopolysaccharide

9.    [CH-07-09] The high lipid content of the cell wall in acid-fast bacteria reduces the permeability to nutrients. How does this affect growth rate and generation time? –

Growth rate is decreased and generation time is increased due to how slowly the bacteria take up nutrients. This is the trade off for the cell wall being difficult to penetrate with antibiotics and disinfectants.

10. [CH-07-10] Why are mycobacteria showing “corded growth” cell arrangements? –

            Mycobacteria have a cord factor, a glycolipid that contributes to the rope or cord like appearance of the bacteria under the microscope.

Chapter 8 - Acid-fast Stain

1.    [CH-08-01] Why are endospores more difficult to stain than vegetative cells? –

Endospores have a cortex composed of peptidoglycan, dipicolinic acid and calcium ions deposited between the outer and inner membrane of the endospore. In addition, a spore coat is formed on top of the outer membrane making the endospore even harder to penetrate.

2. [CH-08-02] Explain the terms sporulation and germination –

Sporulation is the bacteria process for making an endospore.

Germination is the process of returning an endospore to a vegetative cell.

3. [CH-08-03] List the pathogenic species of Bacillus and Clostridium with indication of the diseases they cause. –

Bacillus anthracis à Anthrax

Clostridium difficile à Antibiotic-associated colitis

Clostridium botulinum à Botulism

Clostridium tetani à Tetanus

Clostridium perfringes à Gas gangrene

4. [CH-08-04] What are dry heat treatment and autoclave conditions required to kill endospores? –

Autoclave conditions à 121ºC at 15psi for 20mins

Dry heat à 170ºC for 2hrs or 200º for 1.5hrs

5. [CH-08-05] Why are endospores so resistant to drying, heat, chemicals, and radiation? –

The endospores comprised of an outer protein coat, outer membrane, cortex, and inner membrane. These layers are barriers to chemicals and heat applied to the endospore as well as containers for what water does remain in the cell’s dehydrated state. Proteins tightly bounded to the DNA prevent any radioactive damage.

6. [CH-08-06] How long can endospores exist and still retain the ability to germinate?

Endospores can exist for thousands of years à There are stories of endospores taken from Egyptian tombs and from ancient amber that germinated when supplied with nutrients.

7. [CH-08-07] What is the purpose of commercial sterilization of cans? What are the conditions for commercial sterilization of cans? –

Preventing botulism by killing the endospores of Clostridium botulinum.

Conditions are 121ºC for 3mins at 15psi.

8. [CH-08-08] Broth prepared from yeast, meat or vegetative matter is sterilized by boiling. Broth from hay, however, frequently shows bacterial growth despite boiling. Explain why –

Broth from active organic materials or materials with lots of nutrients are more likely to consist of vegetative bacteria since the environments provide room for a lot of growth. However, hay and soil are more likely to consist of endospores since nutrients in those sources are scarcer.

9. [CH-08-09] Is canned food sterile? Yes, No. Explain your answer –

No, canned food is not considered sterile because thermophilic bacteria can survive autoclaving at 121ºC at 15 psi for 3mins. However, at room temperature thermophiles cannot grow, making canned food safe for general consumption.

10. [CH-08-10] How can endospores be eliminated from air? How can endospores be eliminated from bedding?

Eliminated from air: HEPA filter filtration of air.

Eliminated from bedding: ethylene oxide treatment.

Microbiology: Lab Review Chapter 4,5,6

Slightly late with this post...

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.