Cumulative Lab Final PDF

# Cumulative Lab ## Fungi: Molds & Yeasts - Molds are fungi that form colonies composed of microscopic, rounded, intertwining filaments called hyphae. - There are two different structural types of hyphae in fungi. - One type is characterized by individual cells that are separated by the form...

# Cumulative Lab ## Fungi: Molds & Yeasts - Molds are fungi that form colonies composed of microscopic, rounded, intertwining filaments called hyphae. - There are two different structural types of hyphae in fungi. - One type is characterized by individual cells that are separated by the formation of septa or cross-walls, with each cell having a typical eukaryotic structure. - Pores in septa allow streaming of cytoplasm between cells. - Growth of a mold colony occurs at ends of hyphae, called atypical tips. - Hyphae cover the surface of a substrate to form a branching, filamentous network called a mycelium. - A visible mold colony is a mat formed by the hyphae that make up this substrate mycelium. ### Molds - Non-septate hyphae - Septate hyphae ### Yeasts - Blastospore - Pseudohyphae ### Structural Differences Between Molds & Yeasts ## Yeasts - Yeasts are fungi that do not ordinarily form hyphae. - The primary mode of reproduction for yeasts, ie Saccharomyces cerevisiae is the process of budding, whereby a bud forms and eventually separates from the main cell to form a new yeast cell. - Yeasts are also responsible for infections in humans - Candida albicans causes infections in various body sites. - This organism can infect the mouth and tongue, especially in newborns, to cause thrush, causing the tissue to have a chalky appearance. - This often results when Candida is introduced from the mother's vaginal tract during birth, and because newborns initially lack a normal flora in the mouth, Candida can establish a presence and thrive. ## Cumulative Portion of Final Lab Quiz 1. **Process for disinfection of work surfaces.** - Cleancoat first! Wipe down the desktop with disinfectant (bleach) located at each workstation. - Paper towels used to disinfect your workstation can be disposed of in the regular trash. - When you have completed the lab activity and put away all your equipment, disinfect (bleach) your workstation. - After you have cleaned your workstation, remove your lab coat, then wash hands with antibacterial soap near the sink. 2. **Inoculation** - process of introducing microorganisms, cells of biological material (eg. viruses) into a culture medium, host organism, or environment under controlled conditions to promote growth or study specific interactions. 3. **Media** – nutrient-rich substances that support the growth of microorganisms or cells. They can be liquid, solid, or semi-solid and contain essential nutrients for cultivation and study. Food source. 4. **Culture** – intentional growth of microorganisms in a nutrient-rich medium under controlled conditions. It is used to study, identify or produce microorganisms for research or practical applications. ## Process of Inoculation of an Agar Slant - Inoculating loop is heated until red-hot (7-10 secs) - Remove cap from slant culture, heat mouth (5 secs) - Pick up culture, heat mouth of slant, replace cap - Open sterile agar slant, heat mouth - Slant surface is streaked in zigzag motion, heat mouth, recap - Inoculate loop and return ## Presence of Microorganisms in Nearly Every Environment - **Ubiquitous** ## Types of Culture Media That Are Solid - Nutrient-rich agar - Blood agar - MacConkey agar - Mannitol Salt agar (MSA) - Eosin Methylene Blue (EMB) agar ## Use of Higher-Power Objective Lens - Used to magnify the specimen at a higher level, providing greater detail and resolution. - Ideal for observing fine structures, ie detailed morphology of cells, tissues, or microorganisms. ## Role of Each Reagent in Gram Stain Process - **Primary stain (crystal violet):** Stains all cells purple by binding to cell wall. - **Iodine (mordant):** Forms a crystal violet-iodine complex, enhances dye's binding to peptidoglycan in gram+ cells (not a stain). - **Ethanol (alcohol) Decolorizer:** Removes CV-iodine complex from gram- cells, leaving them colorless, while gram+ cells retain the purple color. - **Counterstain (safranin):** Stains decolorized gram- cells pink/red, while gram+ remain purple ## Determining a Pure Culture by Looking at Image - Determined by observing uniformity in colonies - All colonies should have the same shape, size, color, and texture - Should be no variations or distinct morphologies indicating the presences of multiple microorganisms. - **If these characteristics are consistent across the sample, it is pure.** ## Calculation of Bacterial Counts From Dilution Plate - For 1 ml - count 9's - that's how many zeros to add. - For 0.1ml - count 9's then add an extra zero. - Countable agar plate - contains 30-300 colonies - # of cells on countable plate = (## of colonies / 100) *10^Dilution factor of plate ## Motility is Determined by What Kind of Slide Technique? - **Hanging drop technique:** A drop of liquid culture is suspended under a cover slip in a concave depression slide, allowing observation of live, moving microorganisms. - **Wet mount technique:** A drop of liquid culture is placed on a slide and covered with a coverslip, enabling observation of motility under a microscope ## Positive/Negative Citrate Test Color - **Positive:** Royal blue color (indicator - bromothymol blue) - **Negative:** Green - **Streak needle across surface of slant, then stab needle in the thickest part of agar** ## Urease Test - Color of a Positive Reaction - Turns hot pink/fuchsia – it is positive. - Original broth was salmon/light pink. - No reagent only indicator - phenol red ## Analyzing Catalase Test Results - Add 2 dropperful of H₂O₂ (hydrogen peroxide reagent) to catalase tube. - Formation of bubbles (soda fizzing) is a + result ## Types of Growth on EMB Agar - Dark colonies: Gram-negative bacteria that can ferment lactose - Green metallic colonies: Gram-bacteria that rapidly ferment lactose - E. coli - Pale (white) colonies: Gram- bacteria that cannot ferment Lactose. ## Types of Hemolysis on Blood Agar Plate - Alpha hemolysis (smallest ring around colony) - Beta hemolysis (biggest ring around colony) - Gamma hemolysis (middle) no hemolysis – breakdown of RBC ## For Coagulase Test is Used for What Bacteria - Staphylococcus aureus. ## What is Mannitol-Salt Agar Selective & Differential For? - **Selective:** Gram+ Staphylococcus - **Differential:** S. aureus - yellow color change ## What is Agglutination? - Clumping of particles, typically caused by interaction between antigens and antibodies. - Clumping of bacterial cells when they are exposed to specific antibodies ## The Biggy Agar is Selective for What? - **Selective:** Yeast - Candida (albicans, tropicalis ) - **Differential:** The species of within the genus Candida. ## Enteropluri Test System - Incorporates 15 biochemical tests into a single tube that can be simultaneously inoculated. - The test system can be used to identify gram-negative bacillus or coccobacillus bacteria that are suspected to belong to the Enterobacteriaceae. - Before using this test, an oxidase test should be performed as nearly all Enterobacteriaceae are oxidase-negative. If bacteria is positive oxidase-positive, the test system should not be used. ### Procedure - **Inoculating EnteroPluri tube** - Write intials and unknown # on white label on side of tube. - Place several drops of bleach on a paper towel. - Unscrew both caps from tube and place caps on bleach spot on paper towel. - One end of wire is formed into a loop that will act as handle. Do not touch needle end of tube. - Hold tube like pen, gently scrape a small amount of bacteria from unknown TSA plate. - Grasp loop end of wire and gently twist it and then pull on it to withdraw the wire from tube and inoculate each chamber. - **Inoculating the chambers** - Be sure the tip of wire reaches the last chamber, DO NOT completely remove wire from tube. - Use turning motion, push wire back through all 12 chambers until notch on wire is aligned with opening of tube. - Notch is about 4cm from loop end of wire. - Tip of wire should be visible in citrate compartment. - **Reinserting the wire into the tube.** - Break wire at notch by bending it. - Portion of wire remaining in tube maintains anaerobic conditions. - Essential for fermentation of glucose, production of gas, and decarboxylation of lysine and ornithine. - **Breaking off the loop end of the wire.** - Without touching end of wire, use retained portion of needle to punch holes through thin plastic coverings over small depressions on the sides of last eight compartments (adonitol, lactose, arabinose, glycerol, Voges-Proskauer, dulcitol/PA, urea, and citrate). - These holes will enable anaerobic growth in those eight compartments. - Use wire fragment to poke holes through back wall on chamber with U-shaped depressions. - Do not punch through plastic film on the bottom of the tube. - Put piece of wire in a biohazard disposal bag. - Replace caps at both ends. - Incubate at 37°C for 18 - 24 hrs with Enteropluri tube lying on its flat surface ### Interpreting the Results - Examine each chamber and use info in *Biochemical Reactions of Enteropluri Test section* to determine if positive or negative. If +, circle # on data sheet below its name. - Use a toothpick, probe hole in plastic film covering H2S/indole chamber. Rotate toothpick to enlarge hole, place tooth in biohazard bag. - Add 1-2 drops of Kovac's (indole) reagent to H2S/indole chamber. Red color on surface of medium in 10 secs, test is +, circle # on data sheet. ## Biochemical Reactions of Enteropluri Test - **GLUC = glucose:** - Any degree of yellow is +; orange is - - **GAS = gas production (in glucose chamber):** - Complete separation of wax overly from surface of glucose medium occurs when gas is produced. - **LYS = lysine decarboxylase** - Any degree of purple (acidic) is +; yellow if decarboxylation of lysine does not occur. - **ORN = ornithine Decarboxylase.** - Purple is positive; yellow if decarboxylation of ornithine doesn't occur. - **H2S = H2S production (hydrogen sulfide).** - Black is +; brown should not be considered +. - **IND = indole formation** - After Kovac's reagent, if pink/red, the test is positive. - H2S production and indole formation are located in the same chamber. Do last - **ADON = adonital** - Simple sugars - **LAC = lactose** - **ARA = arabinose** - **SORB =sorbitol** - **DUL = dulcitol** - Any sign of yellow (acidic) is positive. - Orange/red (alkaline) is negative. - **PA = phenylalanine deaminase** - Yellow (acidic) is positive; green (alkaline) is negative. - **DUL and PA in the same chamber, both are either negative (green), or one is positive. Both cannot be positive. ** - **UREA = urea** - Magenta/hot pink (acidic) is positive, yellow (alkaline) is negative. - **CIT = citrate** - Blue (alkaline) is positive, green (acidic) is negative. ## Watering Testing: Membrane Filtration Method - The purpose of the lab is to determine if a sample of water is potable (safe to drink). - Water contaminated with fecal coliforms could potentially make a person sick if consumed. - Fecal coliforms are gram-negative bacteria. - A small number of fecal coliforms in a water sample are not enough to make you sick. - To detect microbes present in small concentrations, a known volume of water is filtered to collect any bacteria present on the surface of a membrane filter. - 100 ml of water is filtered through a vacuum filtration unit. - The filter is transferred to an EMB agar plate. - EMB allows gram- bacteria to grow and inhibits gram+ growth. - Gram-negative can ferment lactose. - Colonies that grow have a dark color or a green metallic sheen. - Coliform concentration = ( # colonies ) / ( 100 ml ) - Units for this water test are “coliform colonies/100ml” - In order for water to be considered potable, it must have less than (<) 1 coliform colony/ml. - (8)(100ml) = 8.0 coliform colonies/100ml – not potable, more than 1 coliform colony - (5)(100ml) = 0.5 coliform colonies/100 ml - potable ## Slide Agglutination Test: Serological Typing - Can be used to identify microbial species by using antibodies that are specific for different microbial antigens. - Solutions of purified antibodies called antisera are used. - Antibodies will react with specific antigens on the surface of bacterial cells, causing cells to agglutinate (cluster together). - Antigens and antibodies = come together (complex) through precipitation or agglutination. - Grainy appearance of clumps are positive. - Coagulase enzyme is usually present. ## Double Gel Immunodiffusion (Ouchterlony Test) - Double gel immunodiffusion (antibody/antigen) test or Ouchterlony test can be used to detect presence of 1 or more antigens in a patient's sample. - Different known antisera (purified antibodies) are added to wells surrounding the center unknown well. - Wells will diffuse, precipitation reaction will occur where antibody and antigen meet. - Liquid diffuses outward from patient unknown. - Swab sample also diffused outwards. - When Ab/Ag complex, they form a white band.