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.