Lab 6

1. Examine your cultures and determine the effective concentration and contact time for Gram negative and Gram positive organisms by completing the chart below.
2. Use "0" to designate no growth.
3. Use "+" to designate visible growth (some turbidity/growth).
4. Use "++" to designate good growth (increased turbidity/growth).

• Gross appearance (microbes' growth and colony morphologies),
• Staining characteristics (Gram reaction, cell shape, and arrangement), and
• Environmental Requirements

• 'Dichotomous' means having two categories or classifications.
• A dichotomous key is based on successive questions.
• In microbiology, it is used to identify an organism.
• Each question has two possible answers.
• Each of the two possible answers directs the individual in a completely different direction to different questions
• Eventually, no more questions can be asked, and the identity of the organism is revealed.
• In microbiology, the first question to be asked is always 'what is the Gram stain reaction?'
• If the answer is 'Gram positive,' then the individual will follow the series of questions that are asked of all organisms staining Gram positive.
• If the answer is 'Gram negative,' then the individual will follow the series of questions that are asked of all organisms staining Gram negative.

In today's lab, we are going to look at the different ways that questions about environmental, or growth, requirements can be asked of bacteria. Recognize that it is not standard practice for microbiologists to perform all of these tests when faced with an unknown. Only those tests that are appropriate, based on the dichotomous key and the microbiologist's interpretations, are utilized. However, because the purpose of this lab is to become familiar with some of these tests, we will look at a variety of examples.

 Aerobic or Anaerobic?

 Microbiologists do not spend a great deal of time in the lab analyzing exactly how much oxygen an organism requires. Rather, they focus on two things:

• Can the bacteria grow in an environment absent of oxygen?
• If no, these organisms are strictly aerobic.
• If yes, can they also grow in an environment filled with oxygen?
• If the answer is no, these organisms are strictly anaerobic and require special growing conditions; anaerobe jars are used to form a completely oxygen-free environment.

• If the answer is yes, then this means that the organisms can utilize oxygen and will grow quicker and better in an oxygen-rich environment.
• This is because many more ATP molecules are produced as a result of aerobic respiration than anaerobic respiration.
• The more ATP, the more energy available to the organisms for developing, growing, and reproducing.
• Think of ATP as money in the bank. Bacteria that can undergo aerobic respiration have much more money (energy) in the bank than bacteria that can only undergo anaerobic respiration. The rich bugs get fatter quicker.
• Can the bacteria grow in an environment rich in oxygen?
• If no, these organisms are strictly anaerobic and require special growing conditions and anaerobe jars are used to form a completely oxygen-free environment (see above).
• If yes, can they also grow in an environment lacking oxygen?
• If the answer is no, these organisms are strictly aerobic.
• If the answer is yes, this means the organisms can grow in either environment but will do better in an aerobic environment (see above).

• Contains 5% sheep's blood.
• Is considered a basic medium in that almost any type of bacteria can grow on it.
• Is considered an enrichment medium because it is designed to promote the growth of fastidious (picky) bacteria that require specific growth factors not usually found in nutrient media.
• Is also considered to be a differential medium because of its ability to distinguish types of red blood cell hemolysis that are caused by the bacterial colonies.
• Alpha hemolysis causes an area of green to appear in the agar surrounding the bacterial colonies.
• This is typical of Streptococcus pneumoniae.
• Beta hemolysis causes a clear area to appear in the agar surrounding the bacterial colonies.
• This is typical of Streptococcus pyogenes.
• Gamma hemolysis occurs when there is no hemolysis present in the agar surrounding the bacterial colonies.
• This is typical of Enterococcus species.




The blood agar plate, below, has been divided into three sections.

The section labeled "b" has been colonized by beta-hemolytic organisms. Note the clear area where complete hemolysis of the red blood cells occurred by the action of the bacterial colonies.

 The section labeled "a" has been colonized by alpha-hemolytic organisms. Rather than a cleared area, note the green, incompletely hemolyzed area that occurred due to the action of the bacterial colonies.

 The third section, labeled "g" is hard to see. That is because the red blood cells in the agar have not been damaged in any way by the bacterial colonies.

 



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Mannitol Salt Agar

• This medium contains a high salt concentration that inhibits the growth of non-halophiles and encourages the growth of halophiles.
• Thus, this is a selective medium.
• Most Staphylococcus species will grow here.
• This medium also contains mannitol, which is a sugar that is fermented by some organisms, and a pH indicator that causes the agar to turn yellow when acid is present.
• Thus, this is also a differential medium, as it distinguishes between fermenters of mannitol and non-fermenters of mannitol.
• S. aureus ferments mannitol; S. epidermidis does not ferment mannitol.

 



 

 

Can you tell which of the two mannitol salt plates right contains the pathogenic halophile, S. aureus?

 

 

 

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Phenylethyl Alcohol Agar

• This medium contains phenylethyl, an inhibitor of DNA synthesis in aerobic, Gram negative organisms.
• Thus, it is selective in that it selects OUT aerobic, Gram negative organisms and allows Gram positive and anaerobic Gram negative organisms to grow.



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Bile Esculin Agar

• This agar contains bile, which inhibits Gram positive organisms, other than Enterococcus.
• Sometimes, sodium azide, which inhibits Gram negative organisms, is added.
• The organisms of greatest clinical interest within this genus are E. faecalis, E. faecium, E. durans, and E. avium.
• These organisms are responsible for causing subacute endocarditis, pyelonephritis, urinary tract infections, meningitis, and biliary infections.
• They are considered to be normal flora, when they are found within the intestinal tract.
• Thus, bile esculin agar is selective in that it selects OUT all Gram positive organisms except Enterococcus.
• It is even further selective when sodium azide is added, as all Gram negative organisms are selected OUT as well.
• This agar also contains esculin, which is easily broken down (hydrolyzed) by many organisms. However, in the presence of bile, only a few organism are capable of hydrolyzing it, including Enterococcus.
• By including a color indicator called ferric citrate in the medium, a microbiologist can immediately recognize that hydrolysis of esculin has occurred, because the medium turns black.
• Thus, bile esculin agar is also differential, in that it differentiates those organisms capable of hydrolyzing esculin in the presence of bile from those that are not.


 

 

 

Which one of these two tubes of bile esculin agar contains an organism capable of breaking down esculin in the presence of bile? When you see this type of reaction, think Enterococcus.

 

 

 

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MacConkey Agar

• Due to the large amount of normal flora present in feces, it becomes difficult to separate out and identify any potential pathogens.
• There are several potential pathogens in feces, Salmonella, Shigella, Yersinia, enteropathic E. coli, S. aureus, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Bacillus species when Bacillus is associated with food poisoning. When present in significant amounts, yeast (fungi) are also considered pathogenic.
• Normal enteric flora are mostly facultatively anaerobic Gram negative rods.
• Of these, one group ferments lactose and is called collectively the "coliforms" and includes mostly non-pathogens, when contained within the intestines.
• Coliforms are pathogens when they infect wounds, the urinary tract, and other tissues.
• The other group does not ferment lactose and includes GI pathogens such as Shigella and Salmonella.
• Generally speaking, the distinction between a lactose fermenter and a non-lactose fermenter is an important one because most GI pathogens will be lactose non-fermenters.
• MacConkey agar contains bile salts and crystal violet, which inhibit the growth of Gram positive bacteria
• Thus, it is selective.
• Sometimes, the crystal violet is left out, so that S. aureus, if present, will grow.
• MacConkey agar also quickly distinguishes the lactose fermenters from the non-fermenters, because it contains a pH indicator that is colorless under conditions close to neutral and red under acidic conditions.
• Thus, coliforms turn red.
• Any organism that remains colorless on MacConkey's requires further testing, as it could be pathogenic, if the source is fecal.
• If the source is other than fecal matter, then any organism growing on MacConkey's requires further testing.
• Because of the MacConkey's ability to distinguish lactose fermenters from non-fermenters, it is also differential.



Most of this MacConkey plate is covered with red (pink) coliforms. However, there are some suspicious clear colonies that require further testing in order to rule out pathogens such as Salmonella or Shigella.

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Hektoen Enteric (HE) Agar

• This is used on fecal specimens for the isolation of enteric pathogenic bacteria.
• It contains high concentrations of bile salts and inhibits the growth of Gram positive organisms and some enteric non-pathogens while encouraging the growth of enteric pathogens such as Shigella and Salmonella species.
• Thus, it is a selective medium.
• It also contains three carbohydrates, lactose, sucrose, and salicin, and two dyes, bromthymol blue and acid fuchsin.
• Bromthymol blue turns yellow under acidic conditions and blue under alkaline conditions.
• Non-pathogenic Gram negatives are lactose fermenters and their colonies appear orange.
• Pathogens such as Shigella and Salmonella species are lactose non-fermenters and their colonies appear green (same color as medium).
• In addition, this medium contains thiosulfate and ferric ion.
• Those organisms that can reduce thiosulfate to H₂S produce a black coloration that is due to the combination of H₂S with the ferric ion.
• This further differentiates organisms into two groups, those that can reduce sulfur and those that cannot reduce sulfur
• This helps distinguish potential pathogens from non-pathogens
• Non-pathogenic, lactose non-fermenters will reduce sulfur (e.g., P. mirabilis, a common enteric non-pathogen).
• Some pathogens, such as Salmonella species, may or may not reduce sulfur. They appear blue or blue-green with black centers, if they reduce sulfur.
• Other pathogens, such as Shigella species, never reduce sulfur.

 



 

 

 

 

 

 

 

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Take Out Food for the Brain:

"Disease eradication" is a 20th century idea. It has happened only once. In 1977, smallpox was eradicated, consigned to test tubes in a few laboratories around the world. This project took a massive international cooperative effort and was successful due to a variety of factors, including the fact that the smallpox virus has no animal reservoir.

Vaccination programs in the United States did not really take flight until 1949, when a combined diphtheria and tetanus toxoid and pertussis vaccine was licensed. The introduction of the Salk poliovirus vaccine in 1955 led to federal funding of state and local childhood vaccination programs. American children's rights to good preventive health care were further protected by the passage of the Vaccination Assistance Act in 1962. This legislation supports the purchase and administration of a broad range of vaccines for children.

Today, vaccinations protect children from ten serious diseases, most of which are spread through droplet transmission. These diseases are:

1. Diptheria
2. Tetanus
3. Pertussis
4. Polio
5. Measles
6. Mumps
7. Rubella
8. Haemophilus influenzae type b disease
9. Hepatitis B (blood/body fluids contact)
10. Varicella

Our government feels so strongly about vaccinating children that children must demonstrate proof of vaccination or disease before being permitted to begin school. There are extenuating circumstances that allow for waivers from this requirement in individual cases.

Since the 1950s, the incidence of these infectious diseases has dropped precipitously. However, there is a coalition of citizens that feels vaccines are unnecessary, especially since "these diseases are not around anymore," and may even contribute to disorders such as acquired immune deficiency syndrome (AIDS) and sudden infant death syndrome (SIDS), and behavioral problems.

Do vaccines prevent disease or do they contribute to disease? You tell me.



  Take Home Thought

Why Vaccinate?







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