Bio 2230 Microbiology Fall 2024 Lab Final Review PDF

Bio 2230: Microbiology Fall 2024 Laboratory Final Review  Ubiquity of Microorganisms Free living → non pathogenic Living in/on a host → can be pathogenic, parasites Commensals or even mutualistic can become opportunistic pathogens Reservoir → source of infection  Stai...

Bio 2230: Microbiology Fall 2024 Laboratory Final Review  Ubiquity of Microorganisms Free living → non pathogenic Living in/on a host → can be pathogenic, parasites Commensals or even mutualistic can become opportunistic pathogens Reservoir → source of infection  Staining Techniques Simple stains = the bacterial smear is stained with a single reagent, which produces a distinctive contrast between the organism and its background Differential stains = allow you to differentiate between different organisms based on the chemical composition of cellular components. It requires at least 3 reagents- a primary stain, decolorizing agent, and counterstain – Gram stain (most bacteria) – Endospore stain (spore formers, such as Bacillus and Clostridium)  Examples of Differential Stains A Gram stain of Staphylococcus aureus and E. coli An acid-fast stain of Mycobacterium smegmatis showing Gram-positive cocci (purple) and Gram- (A-F positive, red) and E. coli (A-F negative, blue) negative bacilli (pink) An endospore stain of Bacillus subtilis showing the endospores as green and the vegetative cells as red  Evaluation of Media Components of media – Proteins – Carbohydrates – Lipids – Nucleic acids (needed for cell growth) Defined and undefined media – Defined media has known quantities of all ingredients – Undefined media has complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions Some characteristics of microbial growth – Fastidious- has a complex nutritional requirement – Non fastidious  Blood Agar Blood agar is an enrichment and differential media Enrichment – blood is the enrichment ingredient Differential – differential for types of hemolysis (the breakdown of RBCs) – Exotoxins (Hemolysins)→ destroy RBCs and hemoglobin Types of hemolysis Beta-hemolysis: complete destruction of RBCs – Staphylococcus aureus – Streptococcus pyogenes Streptococcus pyogenes on blood agar, – Bacillus sp. demonstrating beta-hemolysis Alpha-hemolysis: partial destruction of RBCs – Streptococcus pneumoniae Gamma-hemolysis: no hemolysis – Staphylococcus epidermidis Staphylococcus epidermidis on blood Streptococcus pneumoniae on blood agar, agar, demonstrating gamma hemolysis demostrating alpha-hemolysis Selective Media for Isolation of Gram-Positive Cocci Mannitol salt agar (MSA) – Specifically selective for halophilic bacteria such as Staphylococcus spp. – Differential for mannitol fermentation Mannitol Salt Agar A) Staphylococcus aureus, growth, manntiol positive B) Staphylococcus epidermidis, growth, mannitol negative C) E. coli, no growth, inhibited  Selective Media for Isolation of Gram-Negative Rods (used primarily for members of the family Enterobacteriaceae) MacConkey Agar (MAC) - bile salt, crystal violet, lactose, neutral red dye – Selective – Gram negative – Differential - Lactose 1 Examples of organisms on media 1) E. coli - extremely strong lactose fermenter 4 2 2) Enterobacter aerogenes - slow lactose fermenter 3) Salmonella typhimurium – lactose negative, H2S positive 4) Shigella flexneri – lactose negative 3 MAC agar  Aerotolerance Classification of organisms based on oxygen needs 1.Obligate aerobe (Alcaligenes faecalis) 2.Obligate anaerobe (Clostridium spp.) 3.Facultative anaerobe (Enterobacteriaceae, Staphylococcus epidermidis) 4.Microaerophile (Campylobacter spp.) 5.Aerotolerant anaerobe 6.Capnophile (thrive in the presence of high concentrations of carbon dioxide; i.e. Campylobacter) Ways to investigate the aerotolerance spectrum Media with oxygen gradient: Thioglycollate broth medium, BHI shake-tube method, or agar deep tubes Standard media under aerobic versus anaerobic incubation (standard aerobic versus anaerobic jar with GasPak) Clostridium sp., an obligate anaerobe species  Catalase Test Continued Application: Differentiates Gram-positive cocci Members of the Micrococcaceae family (Staphylococcus spp., Micrococcus spp., Kocuria spp.) are catalase positive Members of the Streptococcaceae family (Streptococcus spp., Enterococcus spp.) are catalase negative 2H2O2 catalase 2H2O + O2↑ Hydrogen Peroxide bubbles 1 2 Slide Catalase Test 1) Staphylococcus epidermidis, catalase-positive 2) Streptococcus pyogenes, catalase-negative  SIM Medium Application: Test for S-Reduction (H2S production), Indole production (from Tryptophan) and Motility. Often used to differentiate between members of the Enterobacteriaceae. Allows multiple biochemical determinations with a single inoculation – Sulfer-Reduction: black precipitate FeS (H2S production) – Indole Production: Kovac’s Reagent is added, indole production and reagent produce a red color – Motility: the reduced agar concentration (semisolid medium) allows for growth throughout tube if the organism is motile, and growth only along the stab line if it is nonmotile Left: H2S neg; Right: H2S pos Left: indole neg; Right: indole pos Left: nonmotile; Right: motile  Carbohydrate Fermentation Tests Glucose is a monosaccharide that is fermented to produce pyruvate and other end products – If an organism can ferment another carbohydrate besides glucose, it will first break down the specific polysaccharide into glucose monomers and ferment the glucose End products of fermentation are dependent on substrate fermented and species involved – Variety of organic acids – Alcohols – H2 – CO2 Phenol Red Broth is a differential test medium for carbohydrate fermentation Phenol red carbohydrate broth – Phenol red is prepared as a base – A specific carbohydrate is added, along with phenol red as indicator dye – A Durham tube can also be added to check for gas production  Coagulase Test Application: Differentiate Staphylococcus aureus from other Gram positive cocci Coagulase forms protective fibrin barriers around cells. Bound coagulase (cell wall associated protein) and free coagulase (enzyme secreted extracellularly) Coagulase Tube test: inoculation of rabbit plasma with organism; coagulation of rabbit plasma demonstrates the presence of coagulase Coagulase Tube Test Top: Staphylococcus aureus, coagulase-positive Bottom: Staphylococcus epidermidis, coagulase-negative  Coagulase Test Continued Questions to consider: What is coagulase? Why is it useful for an organism to be able to produce coagulase? Bacterial Unknown Identification How would you go about identifying an unknown organism? Obtain pure cultures!! Gram stain to determine Gram reaction and shape/arrangement Utilize flowcharts and dichotomous keys Perform necessary biochemical tests appropriate for your organism – Carbohydrate fermentation tests – Agglutination tests – Nitrate test – Antibiotic susceptibility tests – Hemolysis – Etc.  Identification of Enterobacteriacae The Enterobacteriaceae comprise a family of Gram-negative that inhabit the human intestinal tract The majority of species share the following major characteristics – Gram-negative rods – Growth on MacConkey agar – Facultative anaerobes – Ferment glucose – Oxidase negative – Most are catalase positive – Most reduce nitrate (NO3) to nitrite (NO2)  Identification of Enterobacteriaceae Continued Once you have established that an unknown organism belongs to the Enterobacteriaceae family, the IMViC series of tests is the primary series of tests used for identification of the organism – Indole – Methly Red – Voges-Proskauer – Citrate A Gram stain showing Gram-negative rods Based on the IMViC results, additional tests are performed to identify the species of Enterobactericeae  Identification of Enterobacteriaceae Continued IMViC series Indole Test after addition of Methly Red Test after addition Voges-Proskauer Test after Citrate Utilization Test Kovac’s reagent of Methyl Red reagent addition of VP A and VP B reagents Left: citrate-positive Left: indole-negative Left: MR-positive Left: VP-negative Center: citrate-negative Right: indole-positive Right: MR-negative Right: VP-positive Right: uninoculated control Identification of Gram-positive Cocci Gram-positive cocci are common inhabitants of skin and mucous membranes There are 5 main genera of GPC, which are divided into 2 groups based on their catalase test result – Gram-positive cocci, Catalase-positive Staphylococcus Kocuria Micrococcus A Gram-stain showing Gram-positive cocci in a staphylococcus arrangement – Gram-positive cocci, Catalase-negative Streptococcus Enterococcus A Gram-stain showing Gram-positive cocci in a streptococcus arrangement  Identification of Gram-positive Rods GPRs have environmental, industrial, and clinical importance There are 5 main genera of GPRs, separated into – Bacillus – Clostridium – Corynebacterium – Lactobacillus – Mycobacterium (also acid-fast positive) A Gram-stain showing Gram-positive rods A Gram-stain showing Gram-positive rods with spores  ELISA Test Enzyme Linked Immunosorbent Assay 1 wash 2 wash Steps of ELISA Procedure 1. Add purified ANTIGEN, incubate, then wash 2. Add patient samples (containing primary antibody if the patient is wash 4 Read result positive) and controls to their 3 individual wells, incubate, then wash 3. Add secondary antibody (which is conjugated to an enzyme), incubate, then wash 4. Add substrate, incubate, then read results A color change is a positive result No color change is negative