3-10 endospore lab questions

WHAT DOES A POSITIVE RESULT FOR THE SPORE STAIN INDICATE ABOUT THE ORGANISMS? WHAT DOES A NEGATIVE RESULT INDICATE?

A positive result indicates that the organism forms spores. The greatly reduces the number of possible organisms. A negative result shows that the organism does not form pores under the growth conditions, and possibly at all.

THEORY:

An endospore is a dormant form of the bacterium that allows it to survive poor environmental conditions. Spores are resistant to heat and chemicals because of a tough outer covering made of the protein keratin, which also resists staining so extreme measures must be taken to stain the spore. This this stain (Schaeffer-Fulton) a primary stain of malachite green is forced into the spore by steaming the bacterial emulsion. It could also be left on the slide for 15 minutes or more to stain the spores. Malachite green is water soluble and has a low affinity for cellular materials, so vegetative cells and spore mother cells can be decolorized with water and counter stained with safranin. Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle. Spores also may be differentiated based on shape-either spherical or elliptical - a size relative to the cell. See page 117 in the lab manual for pictures. Culture age can affect sporulation. Bacteria capable of producing spores don't do so uniformly during their culture's growth. Sporulation is done in response to nutrient depletion, so is characteristic of older cultures.

WHY IS STEAM APPLIED IN THIS ENDOSPORE STAIN?

An endospore must be provided with the favorable condition to loosen the layers of endospore. Appropriate conditions include moisture. So steam allows the spore to begin to be rehydrated, we are fooling it; provide favorable conditions to the endospore facilitates effective stain penetration. By staining with malachite green the outer endospore cover appears green and the inner cell appears pink when stained with the counter stain safranin.

IF YOUR SAMPLES CONTAIN ENDOSPORES, WHAT WILL YOU EXPECT TO OBSERVE ON YOUR SLIDE? WHAT ABOUT IF YOU ORGANISM DOES NOT PRODUCE ENDOSPORES?

As Malachite Green is a water soluble stain, it washes out of vegetative cells, but not out of the endospores. Safranin is then applied, which stains the vegetative cells reddish (it is unable to get into the endospores). If the species produces endospores, one will likely see roundish green endospores and reddish rods. If the species does not produce endospores, one will only see reddish vegetative cells.

WHICH GENERA OF BACTERIA CONTAIN SPORES AND WHAT ARE THEIR SHAPES? ARE THESE GRAM POS. OR GAM NEG?

Bacillus - most are soil, freshwater or marine saprophytes and a few are pathogens (ie B. anthracis, the causal agent of anthrax) Clostridium - most are soil or aquatic saprophytes or inhabitants of human intestines, but 4 pathogens are fairly well known - C. tetani (tetanus), C. botulinum (botulism), C. perfringens (gas gangrene), and C. difficile (pseudo-membranous colitis). Endospores are dormant, non-reproductive and enzymatically inert forms of bacterial vegetative cells. Endosporeproducing bacteria are more conventionally termed "sporulating bacteria". Sporulating bacteria are derived from the Firmicute phylum and mainly consist of Gram positive bacteria. These endospore-forming bacteria belong to the Bacillus and Clostridium genus

WHY IS IT NOT NECESSARY TO INCLUDE A NEGATIVE CONTROL FOR THIS STAIN PROCEDURE?

Endospore stain is a differential staining procedure that allows to see both spores and vegetative cells, thus including a separate control consisting of only vegetative cells is not required.

EXPLAIN HOW ENDOSPORES ARE DIFFERENT FROM INCLUSIONS.

Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins. Endospores are defined earlier in this paper.

WHAT IS AN ENDOSPORE? WHY ARE SPORES SO RESISTANT TO ENVIRONMENTAL CONDITIONS? ARE THEY USED FOR REPRODUCTION? \

Microorganisms sense and adapt to changes in their environment. When favored nutrients are exhausted, some bacteria may become motile to seek out nutrients, or they may produce enzymes to exploit alternative resources. One example of an extreme survival strategy employed by certain low G+C Gram-positive bacteria is the formation of endospores. This complex developmental process is often initiated in response to nutrient deprivation. It allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress. Endospores can survive environmental assaults that would normally kill the bacterium. These stresses include high temperature, high UV irradiation, desiccation, chemical damage and enzymatic destruction. The extraordinary resistance properties of endospores make them of particular importance because they are not readily killed by many antimicrobial treatments. A variety of different microorganisms form "spores" or "cysts", but the endospores of low G+C Grampositive bacteria are by far the most resistant to harsh conditions.

APPLICATION:

Only a few genera produce spores. Among them are Bacillus and Clostridium.

DESCRIBE THE APPEARANCE OF A SAMPLE THAT CONTAINS ENDOSPORES IF YOU STAINED THE SAMPLE USING THE GRAM STAIN METHOD. WHAT COLOR SHOULD THE "MOTHER-CELLS" BE STAINED?

Only some gram positive bacteria form endospores: low G+C Gram-positive bacteria Bacillus and Clostridium. So, one will expect the gram stain to show purple stained cells.

GIVE 3 EXAMPLES OD DISEASES CAUSED BY ENDOSPORES.

Some spore formers are pathogens of animals, usually due to the production of powerful toxins. Bacillus anthracis causes anthrax, a disease of domestic animals (cattle, sheep, etc.), which may be transmitted to humans. Bacillus cereus causes food poisoning. Clostridium botulimum causes botulism, a form of food poisoning Clostridium tetani is the agent of tetanus. Clostridium perfringens causes food poisoning, anaerobic wound infections and gas gangrene Clostridium difficile causes a severe form of colitis called pseudomembranous colitis. Whenever the spore-formers act as pathogens, it is not uncommon or surprising that their spores are somehow involved in transmission or survival of the organism between hosts.

WHAT METHOD DOES THE LAB TECHNICIAN USE TO ELIMINATE ENDOSPORES FROM LAB GLASSWARE AND WHY IS IT EFFECTIVE?

While significantly resistant to heat and radiation, endospores can be destroyed by burning or by autoclaving at a temperature exceeding the boiling point of water, 100 °C. Endospores are able to survive at 100 °C for hours, although the longer the number of hours the fewer that will survive. An indirect way to destroy them is to place them in an environment that reactivates them to their vegetative state. They will germinate within a day or two with the right environmental conditions, and then the vegetative cells, not as hardy as endospores, can be straightforwardly destroyed.

WHY USE OLDER CULTURES FOR THIS STAIN?

Young cultures of spore-forming microbes may not demonstrate any endospores because the vegetative cells may not have been subjected to sufficient stress to stimulate sporulation.

WHY DOES THE EXERCISE CALL FOR AN OLDER CULTURE OF BACILLUS?

The resilience of an endospore can be explained in part by its unique cellular structure. The outer proteinaceous coat surrounding the spore provides much of the chemical and enzymatic resistance. Beneath the coat resides 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. A germ cell wall resides under the cortex. This layer of peptidoglycan will become the cell wall of the bacterium after the endospore germinates. The inner membrane, under the germ cell wall, is a major permeability barrier against several potentially damaging chemicals. The center of the endospore, the core, exists in a very dehydrated state and houses the cell's DNA, ribosomes and large amounts of dipicolinic acid. This endospore-specific chemical can comprise up to 10% of the spore's dry weight and appears to play a role in maintaining spore dormancy. Small acid-soluble proteins (SASPs) are also only found in endospores. These proteins tightly bind and condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals. Other species-specific structures and chemicals associated with endospores include stalks, toxin crystals, or an additional outer glycoprotein layer called the exosporium.