Lab 8 Endospore Stains Microbiology

Bacillus characteristics

-gram-positive -obligate aerobes -endospore formers -highly motile -catalase positive - live in soil

Dormant to vetetative

1. Activation- presence of nutrients and other triggers like heat. All factors work together to make sure all needs are met. 2. Germination- Breaking of dormant stage marked by swelling and rupture of the spore coat, loss of spore components and increase of metabolic activity. 3. Outgrowth- New components are made, and an active bacterium emerges from the remains of the spore.

Endospores have

1. Calcium. Calcium helps keep it dry so no metabolic activity occurs and it is not sensitive to environmental conditions. 2. Dipicolinic acid. Dipicolinic acid to make it insensitive to the environment. 3. Small acid soluble DNA binding proteins. DNA binding proteins saturate the spore coat and make it more resistant. 4. Keratin in exosporium. Keratin keeps it waterproof for extra protection.

Endospore stain technique

1. Prepare 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. 5. Place the slide on the beaker of boiling water and steam for 10 minutes, adding additional stain as needed. 6. Remove the slide, add a drop a malachite green, and allow it to cool. 7. Remove the paper towel. Discard in stain bin, not the sink (toxic). 8. Flood the slide with Safranin for 2 minutes and then wash with water. 9. Blot dry with bibulous paper. 10. The endospores stain blue/green while the vegetative cells stain red. Spores can be stained within vegetative cells or outside of the cell.

dyes used

2 basic dyes to stain the negatively charged cell. malachite green steamed to color spores. safranin counterstain to see cell.

Bacterial endospore formation process

A Gram Positive bacteria (Bacilli shape or Sporosarcina coccus species) stressed by environmental conditions like lack of nutrients or temperature changes 1. replicate DNA, 2. grow a spore septum and plasma membrane to separate the new DNA, forming the forespore. 3. grows peptidoglycan and a spore coat 4. is released from the disintegrating cell. Germination returns spore to an active/vegetative state when several environmental trigger conditions are all met. The spore coat ruptures and swells, and endogenous enzymes break it down, allowing water to enter and metabolic activity to resume. In outgrowth, the new components are made by enzymes, and an active bacterium emerges from the remains of the spore.

Endospore

A thick-walled protective dormant spore that forms inside a gram positive bacterial cell and resists harsh conditions when released. It is metabolically inactive. It is difficult for control agents to penetrate the spore coat.

What is an endospore

A tough, nonreproductive structure that helps the bacteria survive times of stress.

Is there an indication of endospore formation in Gram stain?

Clear spots or lighter color spots might indicate endospore or inclusions.

oligotrophic environment

Contain low nutrient concentrations.microbes become more competitive.Morphology may be affected.They might undergo step by step shutdown except for housekeeping genes.They might put aside critical nutrients for future use and to keep it from the competition.The atmosphere may contribute essential nutrients.They may produce endospores if possible.

In the spore stain procedure, why would you steam malachite green? What would be the result if you failed to steam the stain?

Endospores do not normally stain because the dyes do not penetrate the wall of the endospore. Heating allows the dye to penetrate so the spore can be stained green, so if you did not steam the cell the endospore would not be visible.

what bacteria might survive the high heat of canning process?

Endospores from thermophiles

Clostridium characteristics

Gram positive spore forming obligate anaerobes rods

Why is gram reaction not consistent with endospore forming microbes?

Gram positive form endospores. The tough structure of endospores resists stains. Spores and endospores would not be seen with Gram stain. Gram reaction was not consistent to gram type positive because old cultures have weak cell walls and gram variable reaction can be seen.

Bacillus cereus

Gram positive, rod shaped. Facultative anaerobe. Endospore former. may have granules.

Clostridium sporogenes

Gram positive. obligate anaerobe. Produces oval subterminal endospore. Intestinal normal flora, and also soil.

endospore gram type

Gram positive. A number of Gram-positive bacteria of several genera such as Bacillus and Clostridium (both rod-shaped), Sporosarcina (cocci-shaped) can produce endospores.

What type of bacteria form endospores?

Gram positives only. Bacillus types and one coccus species, Sporosarcina.

Staphylococcus saprophyticus

Gram-positive cocci, clusters. aerobe Not an endospore former. UTIs

Stationary Phase

Growth rate slows and the number of microbial deaths match that of new cells and the population stabilizes.Nutrients get depleted, waste products accumulate, and harmful pH changes can occur. Oxygen being used up and toxic environment increasing. The environment does not support exponential growth. Maybe endospores made at this time. Use mid stationary phase for endospore stain. Shows characteristic morphology and metabolic activity at this time, but also shows the endospores. At about 72 hours.

clinical significance of the placement of the endospore within the cell

Helps identify the type of cell to the genus because the placement is characteristic of the species.

Where do you take a sample from in a colony for endospore stain?

In the center of the CFU. The center has older cells that are more likely to have experienced stress from depletion of nutrients and accumulation of toxins.

Are bacterial spores involved in reproduction?

No, spores are survival structures. Spores help increase chance of survival in stressful environment. The DNA is replicated, spore formation occurs, and the mother cell disintegrates. But the number of cells is not increased.

What is the purpose of using the counter stain, saffranin? What would be the result if you forgot to do this step?

Saffranin dyes the parts of the cell that are not endospores, making this a differential stain so the different cell structures can be seen as a different color. If you did not do this, the cells and structures would be green and endospores would not be seen. A counter stain allows different cell types and different cell structures to be seen.

Endospore placement of Clostridium sporogenes

Terminal endospore with swollen sporangium

Spore positions

central, terminal, subterminal, lateral

Safranin

counterstain for endospore staining

mother cell measurement

disintegrates so hard to measure size accurately

sporangium

enclosure in which spores are formed

Green spots

endospores. Can be free or inside the cell.

can endospores be seen in the Gram stain?

no

malachite green

primary stain for endospore stain

swollen sporangium

seen as swelling on endospore forming cells

spore vs. endospore

spore are dormant survival structures that are not especially heat resistant. endospores are specialized survival structures that generally are more heat resistant.

Endospores can survive

temperature, antibiotics, drying, acids, bases, certain disinfectants, UV radiation, gamma radiation, lysozyme, starvation

location of spore used to

used to identify unknown genus and perhaps the species

Schaeffer-Fulton endospore stain

uses heat to drive the primary stain, malachite green, into the endospore The cooling step traps the stain in, and the counterstain Safranin stains remaining cytoplasm outside the spore red.

Pink cells would be

vegetative cells. But you can't always assume they are. ???

Endospore characteristics

withstand hostile condition, facilitate survival. Have 2 phase life cycle (vegetative, or metabolically active) Impermeable spore coat. High content of calcium and dipicolinic acid. Low water content. Very low or no?? enzyme activity. DNA binding proteins saturate the spore DNA and protect it from heat.

Bacteria that produce endospores are resistant to disinfectants, many chemicals, heat and radiation. What contributes to this resistance?

The spores are resistant to environmental conditions and survive to become vegetative again when conditions improve. They can survive for thousands of years: 1. Metabolic activity is suspended in the spores. The cell is less reactive to its environment. Spore coat has characteristics to protect it to keep the metabolic activity suspended. DNA is stabilized and metabolic activity is suspended, so less reactive to heat and radiation. 2. The tough spore coat does not let chemical control agents through. Can be dormant for centuries, so can be disease agents when they emerge. Never really go away.

What advantage do the endospore formers have in reemerging diseases when compared to the non-endospore formers?

They can survive for thousands of years due to tough keratin coating, calcium to absorb moisture, Acid-soluble DNA binding proteins to protect spore coat and preserve DNA, and dipocolinic acid to resist heat. Their metabolic activity is suppressed without several factors causing activation. Their characteristics make them resistant to chemical and physical destructive agents. Their characteristics are kept intact through the DNA being unaffected. Their slow reproductive rate makes treating them with antibiotics a long process.

Why do bacteria form endospores?

To resist lack of nutrients and harsh environmental conditions like high temperatures, and chemical agents like disinfectants