Lab 8

Question 1

In the spore stain procedure why would you steam malachite green

Answer 1

Steaming allows the hot malachite green to penetrate the spore coat and dye them green. If this is not done, then the dye will not penetrate the spore and it will appear clear

Question 2

What is the purpose of using counter stain Safranin

Answer 2

The counter stains the remaining cytoplasm outside the spore red. If this is not done, only the spores will be seen. This will make your hard to identify the location or position of the spore in the mother cell.

Question 3

What are the different endospore locations that are found in different species

Answer 3

Central, terminal, terminal with swollem sporangium, and subcentral

Question 4

What is the primary function of most Endospores

Answer 4

To ensure the survival of a bacterium through periods of environmental stress

Question 5

Most Endospores are resistant to

Answer 5

Ultra Violet and gamma radiation, desiccation, Lysozyme, temperature, starvation and chemical disinfectants

Question 6

What do bacterial cells do in adaptation to nutrient poor environments

Answer 6

They shut down their metabolic pathways and start making protective proteins

Question 7

What dormant structure do bacteria produce that allows prolong survival

Answer 7

Spores or cysts

Question 8

Are all spores heat tolerant

Answer 8

No

Question 9

What are highly specialized survival structures of bacteria

Answer 9

Endospores

Question 10

What is an endospore

Answer 10

Small, highly resistant, thick-walled, “resting” cells
Produced in response to environmental stressors
Formed when bacterial cells STOP growing
Formed within vegetative cells, non reproductive body

Question 11

What genus of bacteria produce endospores

Answer 11

Bacillus, Clostridium, Desulfotomaculum, Sporosarcina, Sporolactobacillus, Oscillospira, Thermoactinomyces

Question 12

Where are bacillus species found

Answer 12

Obligate aerobes the live in soil

Question 13

Where are Clostridium species found

Answer 13

Obligate anaerobes often found as normal flora in GI tract of animals

Question 14

What is the main substance responsible for the resistance of spores

Answer 14

10% dipicolinic acid

Question 15

How does calcium in spores contribute to their resistance

Answer 15

It helps with resistance of heat and oxidizing agents

Question 16

What saturates the spore coat? Does this contribute to their resistance?

Answer 16

Acid soluble, DNA binding proteins

Yes

Question 17

When are endospores revived

Answer 17

they can be dormant for decades

Question 18

How are dormant spores activated to vegetative cells

Answer 18

3 stages

1. Activation
2. Germination
3. Outgrowth

Question 19

Activation stage

Answer 19

Presence of nutrients and treatments like heat trigger activation

Question 20

Germination

Answer 20

The breaking of the dormant stage, an endospore returns to its vegetative state

Question 21

What triggers germination

Answer 21

Physical or chemical damage to the endospores coat

Question 22

Describe what happens during germination

Answer 22

The endospores enzymes break down the extra layer surrounding the endospore and water enters. The endospore swells and ruptures. Metabolic activity resumes and/or increases.

Question 23

Outgrowth

Answer 23

New components are made and active bacterium emergence from the remains of the spores

Question 24

Is sporulation a form of reproduction

Answer 24

It is not a form of reproduction. One vegetative cell forms a single endospore, which after germination remains 1 cell. This process does not increase the number of cells.

Question 25

Describe the process of sporulation

Answer 25

1. Spore Septum begins to isolate newly replicated DNA and a small portion of cytoplasm 2. Plasma membrane starts to surround dna, cytoplasm 3. Spore septum becomes a double layered membrane that surrounds the chromosome in the cytoplasm; forespore 4. Peptidoglycan layer forms between membranes 5. The spore coat protein forms around the outside membrane of the endospore 6. The original cell is degraded and the endospore is freed from the cell

Question 26

What happens to the water present in the forespore cytoplasm

Answer 26

Most of it is eliminated by the time sporulation is complete

Question 27

Do endospores carry out metabolic reactions

Answer 27

No

Question 28

What protects the Endospore from damage

Answer 28

DPA protect the endospore DNA against damage

Question 29

What does the endospore core contain and why

Answer 29

DNA, small amounts of RNA, ribosomes, enzymes, and a few important small molecules. There essential for resuming metabolism later

Question 30

How do older spores appear under microscope

Answer 30

They are smaller and can appear clear inside the cell

Question 31

How do inclusions or granules appear under microscope

Answer 31

Dark in cell

Question 32

What staining method is used for spore stain

Answer 32

Schaefer-Fulton spore stain method

Question 33

Can endospores stained using typical staining procedures such as gram stains

Answer 33

No, they will appear clear, unstained areas inside cell

Question 34

What benefit does the cooling step have in endospore staining

Answer 34

It traps the stain inside and allows the counter stain to color remaining cytoplasm outside the spore

Question 35

Schaeffer-Fulton procedure

Answer 35

1. Prepare a boiling water bath 2. Prepare a bacterial smear and heat fix 3. Place the slide on a staining rack 4. Place a piece of paper towel over the smear and saturate with malachite green 5. Place slide on beaker of boiling water and steam for 10 minutes. Add stain to prevent drying. 6. Remove slide, add a drop of malachite green and cool. Remove paper towel. 7. Wash with water to remove excess stain. 8. Flood the slide with Safranin for 2 minutes and then wash with water. 9. Blot dry

Question 36

What should be the end result of an endospore stain

Answer 36

The endospores stain blue/green while the vegetative cells stain red. Spores can be stained within vegetative cells or outside the cell.

Question 37

How do you label endospore stain results

Answer 37

Vegetative cell or endospore forming cell

Question 38

Gram stain of Bacillus cereus

Answer 38

It's a gram positive bacteria so it should stain positive. Our results were inconsistent with the gram type. It's stained gram negative probably because it was an old culture. It did not have endospores, but showed possible inclusions instead.

Question 39

What methods of microbial control can endospores withstand

Answer 39

Many chemicals, heat, desiccation, extreme freezing and UV radiation

Question 40

What contributes to endospore resistance

Answer 40

The outer coat made up of 10% dipicolinic acid, calcium, and acid soluble, DNA binding proteins. It provides much of the chemical and enzymatic resistance. Small acid-soluble proteins (SASPs) are tightly bound, condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals. Calcium contributes to heat resistance and oxidizing agents. Beneath is a very thick layer of specialized peptidoglycan called the cortex. Proper cortex formation is needed for dehydration of the spore core, which aids in resistance to high temperature. The inner membrane is a major permeability barrier against several potentially damaging chemicals.  

Question 41

Most endospore forming cells are

Answer 41

Gram positive

Question 42

What advantage do endospore formers have in a re emerging diseases when compared to the non endospore formers

Answer 42

They can survive harsh environments such as extreme heat, lack of water, and exposure to harsh chemicals and radiation. They can lay dormant for thousands of years and reemerge when the environment is adequate for survival.