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Bio 15: Ch 10 Classification of Microorganisms study guide Key terms Ch 10
Taxonomy Culture Clone Strain Morphological Characteristics
Ch 10 questions
1. Be able to explain what are biochemical tests based on?
Biochemical tests are based on the principle of detecting and measuring specific chemical reactions or the presence of certain biomolecules that are indicative of a particular biological condition, microorganism, or physiological state. These tests are used in fields like microbiology, clinical diagnostics, and biochemistry. Here’s how they generally work:
- Enzyme Activity : Many biochemical tests focus on detecting enzymes, which are proteins that catalyze specific chemical reactions. For example, some tests might measure whether an enzyme breaks down a substrate into products, producing a color change or a detectable byproduct.
- Metabolic Pathways : The tests can be designed to detect specific metabolic activities. For example, a bacteria’s ability to ferment sugars, produce gas, or utilize specific nutrients can be detected. This is useful in microbiology for identifying bacterial species.
- Chemical Reactions : In clinical diagnostics, tests can detect changes in the presence or concentration of certain molecules, such as glucose, proteins, or electrolytes in blood or urine. These changes are often the result of underlying health conditions like diabetes, kidney disease, or liver function problems.
- Indicators : Many biochemical tests rely on chemical indicators (such as pH indicators or colorimetric reagents) that change color in response to a specific chemical reaction. This makes it easier to observe and interpret the test results. Examples of biochemical tests include: Catalase Test : Detects the presence of the catalase enzyme in bacteria by observing the breakdown of hydrogen peroxide into water and oxygen. Urease Test : Detects the ability of bacteria to hydrolyze urea into ammonia and carbon dioxide. Glucose Fermentation Test : Tests whether an organism can ferment glucose to produce acid or gas. Overall, these tests are based on the chemical and biological properties of the substances involved, which, when altered or reacted in a specific way, can provide important diagnostic information.
2. Be able to give an example of a differential stain and explain what they are used
for.
A common example of a differential stain is the Gram stain. What is a Differential Stain? A differential stain is a laboratory technique used to differentiate between different types of microorganisms or cell structures based on differences in their physical and chemical properties, particularly their cell wall composition. Unlike simple stains, which only color cells uniformly, differential stains allow for the identification of various cell types, structures, or components by producing different colors or reactions. Example: Gram Stain The Gram stain is one of the most widely used differential staining techniques in microbiology. It distinguishes bacterial species into two broad categories: Gram-positive and Gram-negative , based on differences in their cell wall structure. Gram-positive bacteria : These bacteria have a thick layer of peptidoglycan in their cell walls, which retains the crystal violet stain and appears purple under a microscope. Gram-negative bacteria : These bacteria have a thinner layer of peptidoglycan but possess an additional outer membrane, which prevents the retention of the crystal violet stain. Instead, they retain the counterstain (usually safranin), making them appear pink under a microscope. Steps in the Gram Staining Process:
- Crystal Violet (Primary Stain): The bacterial cells are stained purple.
- Iodine (Mordant): Forms a complex with crystal violet, helping it stay inside the cell wall.
- Alcohol or Acetone (Decolorizer): This step differentiates between Gram-positive and Gram-negative bacteria. It dehydrates the thick peptidoglycan layer of Gram-positive bacteria, trapping the crystal violet inside, but it dissolves the outer membrane of Gram- negative bacteria, allowing the crystal violet to wash out.
- Safranin (Counterstain): Gram-negative bacteria take up the red counterstain, while Gram-positive bacteria remain purple. What Are They Used For? Identification of Bacterial Species : The Gram stain helps in the preliminary identification of bacterial species and guides the selection of appropriate antibiotics for treatment. Gram-positive and Gram-negative bacteria often have different susceptibility to antibiotics. Classifying Bacteria : It serves as the first step in bacterial classification. It distinguishes bacteria into two major groups that can have distinct behaviors and pathogenicity.
Example of Application: HIV Testing ELISA is frequently used in diagnosing HIV (Human Immunodeficiency Virus) infection. During the early stages of HIV infection, the body produces antibodies specific to the virus. By using an ELISA test to detect these antibodies, healthcare providers can diagnose HIV, even before symptoms appear. Procedure : A patient's blood or serum is tested for the presence of antibodies against HIV antigens. If the antibodies are present, the test produces a color change, indicating a positive result. Follow-up : If the ELISA result is positive, it is typically followed by a confirmatory test, such as the Western blot or PCR test, to confirm the diagnosis. Applications of Serological Tests: Infectious Disease Diagnosis : Serological tests are used to detect infections like HIV, hepatitis, syphilis, and COVID-19 (for antibodies). Blood Typing : Serological tests can identify blood group antigens (A, B, AB, O) and Rh factor. Vaccine Monitoring : After vaccination, serological tests can be used to check for the presence of antibodies, which shows if the individual has developed immunity. Autoimmune Disorders : Testing for specific autoantibodies helps diagnose diseases like rheumatoid arthritis or lupus. In summary, serological tests are essential tools in modern diagnostics and immunology, helping to identify infections, assess immune responses, and monitor health conditions.
Review
1. Which of the following organisms are most closely related? Are any two the same species? On what did you base your answer? A and D appear to be most closely related because they have similar GC moles %. No two are the same species. 2. Here is some additional information on the organisms in question 1:
Which of these organisms are most closely related? Compare this answer with your response to review question 1. A and D are most closely related.
3. Use the additional information on the next page to construct a cladogram for some of the organisms used in question 4. What is the purpose of a cladogram? How does your cladogram differ from a dichotomous key for these organisms? The purpose of a cladogram is to show the degree of relatedness between organisms. A dichotomous key can be used for identification but doesn’t show relatedness like the cladogram. Mycoplasma and Escherichia are on one branch in the key, but the cladogram indicates Mycoplasma is more closely related to Clostridium. 4. Use the information in the table below to complete the dichotomous key to these organisms. What is the purpose of a dichotomous key? Look up each genus in Chapter 11, and provide an example of why this organism is of interest to humans. 5. Use the key in the Clinical Focus box on page 306 to identify the gram-negative rod causing pneumonia in a sea otter. It is H2S-positive, indole-negative, and urease-positive. Analysis 1. There is an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infection in a hospital. Which genotyping technique can help in the source detection? To detect the source of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in a hospital, a commonly used genotyping technique is Polymerase Chain Reaction (PCR) ,
Identification of virulence factors and resistance genes, which may influence the clinical outcome of the infection. WGS provides the most comprehensive and precise data for tracing outbreaks and identifying source strains, though it is more expensive and time-consuming than PFGE or PCR. Conclusion: For detecting the source of an MRSA outbreak in a hospital, PFGE and PCR-based methods (like SCCmec typing or spa typing ) are commonly used. However, for more detailed analysis and accurate tracing of the outbreak, whole-genome sequencing (WGS) is becoming an increasingly popular choice. These genotyping techniques help identify related strains, track their spread, and inform infection control measures.
2. Nucleic acid hybridization techniques are highly sensitive in nature and, thus, require meticulous optimizations. In what situation would a Southern blotting experiment for the identification of microorganisms mostly fail? A Southern blotting experiment is used for detecting specific DNA sequences in a sample, and while it's a sensitive technique, there are situations where it may fail, especially in the identification of microorganisms. Situations where Southern blotting might fail in identifying microorganisms: 1. Sequence Variation : o High Genetic Diversity : If the microorganism you are trying to detect has a high degree of genetic variation or polymorphism, the specific DNA sequence you're targeting might not be present or might be too different to bind effectively to the probe. This is especially true for organisms that have many different strains or species with considerable sequence differences in the region of interest. o Mutations : If the microorganism has undergone mutations that change the target sequence, the probe may not bind properly, leading to a failure to detect the microorganism. For instance, in MRSA or other pathogens, genetic variations in specific genes could lead to a failure in identification. 2. DNA Degradation : o Poor DNA Quality : Southern blotting requires intact, high-quality DNA. If the sample DNA is degraded due to improper handling, long storage times, or contamination, the results may be inconclusive or fail entirely. DNA degradation can result in incomplete digestion by restriction enzymes, leading to poor or no hybridization. 3. Low Sensitivity : o Low Abundance of Target DNA : Southern blotting is not as sensitive as other molecular techniques like PCR. If the microorganism's DNA is present in very low amounts in the sample (for example, in clinical samples with a small number
of pathogens), the signal may be too weak to detect, leading to a false negative result. o Suboptimal Hybridization Conditions : Southern blotting requires precise conditions for hybridization (such as temperature, salt concentration, and probe concentration). If the hybridization conditions are not optimized, the probe may not bind effectively, leading to failed detection.
- Incorrect Probe Design : o Non-specific Binding : If the probe is not designed specifically for the microorganism of interest or has a similar sequence to other organisms, it may bind non-specifically to DNA from other sources, resulting in ambiguous or false- positive results. o Insufficient Probe Labeling : If the probe is not labeled with an appropriate marker (e.g., radioactive or chemiluminescent), the signal might be too weak to detect, leading to a failure in identifying the microorganism.
- Incompatible Restriction Enzyme : o Ineffective Digestion : Southern blotting requires the use of restriction enzymes to cut the DNA at specific sites. If the restriction enzyme(s) used do not cut the DNA properly (due to methylation, sequence context, or other factors), this could lead to incomplete digestion, poor resolution of DNA fragments, and failed detection. Conclusion: Southern blotting is a powerful technique, but it can fail in microorganism identification under the following circumstances: High genetic variation or mutations in the microorganism’s DNA. Degraded or poor-quality DNA samples. Low DNA abundance in the sample. Suboptimal hybridization conditions or poorly designed probes. Inadequate restriction enzyme digestion. In such cases, more sensitive or specific techniques, like PCR-based methods (e.g., PCR amplification followed by sequencing), may be preferred, as they can be more sensitive and adaptable to varying genetic sequences. 3. SF medium is a selective medium, developed in the 1940s, to test for fecal contamination of milk and water. Only certain gram-positive cocci can grow in this medium. Why is it named SF? Using this medium, which genus will you culture? ( Hint : Refer to page 301.) The SF medium (Sodium Lauryl Sulfate Broth) is named after Sodium Lauryl Sulfate (SF) , which is the key ingredient in the medium that makes it selective. Sodium Lauryl Sulfate acts as an inhibitor for the growth of most microorganisms, but it allows certain types of bacteria, particularly gram-positive cocci , to grow in the presence of this inhibitory agent.
What Went Wrong in the Diagnosis:
- Initial Misdiagnosis : o The patient’s symptoms of fever, chest pain, and cough, along with the presence of gram-positive cocci in the sputum on the first Gram stain, suggested a typical bacterial pneumonia , and the patient was initially treated for lobar pneumonia with penicillin. o However, when a subsequent Gram stain showed gram-negative rods , the diagnosis was revised, and the patient was switched to ampicillin and gentamicin , which were more appropriate for the new identification of Pantoea agglomerans (a gram-negative rod). This organism, however, was biochemically inactive and not the true pathogen. o The correct pathogen, Yersinia pestis , was eventually identified using fluorescent-antibody staining and phage typing. These diagnostic methods were critical in identifying the plague-causing bacteria. The delay in recognizing Yersinia pestis led to the initial treatment being ineffective, which likely contributed to the patient’s rapid decline.
- Incorrect Initial Therapy : o The initial treatment with penicillin was appropriate for gram-positive cocci (likely Streptococcus pneumoniae or Staphylococcus aureus, common causes of bacterial pneumonia), but Yersinia pestis is gram-negative , and penicillin would not be effective. Ampicillin and gentamicin are better choices for gram-negative rods, but it was too late to prevent severe complications.
- Delayed Correct Diagnosis : o The time between the patient’s admission and the identification of Yersinia pestis allowed the infection to progress unchecked. If the diagnosis had been made earlier, more aggressive and appropriate treatments (like chloramphenicol and tetracycline ) could have been administered sooner, possibly saving the patient's life. The rapid progression of plague can be fatal if not treated promptly with the right antibiotics. Why Did the 220 Contacts Receive Treatment? The 220 individuals (hospital personnel, family members, and co-workers) were likely treated with tetracycline as a prophylactic measure due to the highly contagious nature of Yersinia pestis. Plague can spread from person to person through respiratory droplets, especially if a patient is coughing and the bacteria are aerosolized. Here’s why they were treated:
- Person-to-Person Transmission : o Yersinia pestis can be transmitted by respiratory droplets from infected individuals, particularly in cases of pneumonic plague (which can result from a secondary infection of the lungs in bubonic plague or from inhalation of the bacteria). The patient had a cough, and Yersinia pestis was present in both his sputum and blood , indicating possible aerosolization or direct contact with respiratory secretions.
- Prophylaxis for Close Contacts :
o Close contacts (like family, co-workers, and healthcare workers) are at high risk of contracting the disease. Tetracycline was given as a preventive measure to stop potential outbreaks of pneumonic plague among people who had direct or indirect contact with the infected patient. Prophylactic antibiotics can prevent the disease from taking hold in individuals exposed to the bacteria, especially when administered within 7 days of exposure. How Could His Death Have Been Prevented?
- Faster Identification : o The key to preventing death from plague is early recognition and appropriate antibiotic treatment. A more rapid diagnosis, particularly the use of fluorescent- antibody staining and phage typing early in the patient’s stay, might have led to an earlier diagnosis of Yersinia pestis. This would have allowed for the immediate administration of effective antibiotics like chloramphenicol or tetracycline , which are particularly effective against Yersinia pestis.
- Antibiotic Therapy : o If tetracycline or chloramphenicol had been started promptly after the initial suspicion of Yersinia pestis , the patient might have had a better chance of survival, as these antibiotics are highly effective against plague when administered early.
- Appropriate Isolation : o The patient should have been placed in respiratory isolation to prevent the spread of Yersinia pestis to others in the hospital. The possibility of airborne transmission would necessitate precautions to avoid a wider outbreak, especially in a healthcare setting. Conclusion: The patient had bubonic plague caused by Yersinia pestis. The initial misdiagnosis of pneumonia caused by gram-positive cocci and the subsequent delay in identifying Yersinia pestis led to improper treatment, which contributed to the patient’s death. The 220 contacts were treated with tetracycline as a preventive measure due to the high risk of pneumonic plague transmission through respiratory droplets. Early recognition, appropriate antibiotic therapy, and proper isolation protocols could have significantly improved the chances of survival. 2. A 6-year-old girl was admitted to a hospital with endocarditis. Blood cultures showed a gram-positive, aerobic rod identified by the hospital laboratory as Corynebacterium xerosis. The girl died after 6 weeks of treatment with intravenous penicillin and chloramphenicol. The bacterium was tested by another laboratory and identified as C. diphtheriae. The following test results were obtained by each laboratory:
Using the additional information shown below, create a cladogram for these organisms. Do your two keys differ? Explain why. Which key is more useful for laboratory identification? For classification? Step 1 1 of 4 Dichotomous key for unicellular organisms
Pathogens: Trypanosoma, Trichomonas, Giardia Trypanosoma, Trichomonas, Gi
ardia
Step 2 2 of 4 Dichotomous key using rRNA sequences for the unicellular organisms
