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Bacterial Smear - Allows better visualization of specimens using microscopy. Heat Fixing - kills organisms, keeps organisms stuck to slide, allows the specimen to be stained more easily Aseptic Technique - preparing and handling sterile products in a manner that prevents microbial contamination Gram Staining Procedure - 1. Heat fix bacterial smear 2. Cover with Crystal Violet (Primary Stain) and wait 60 seconds 3. Rinse slide with distilled water until stain is removed 4. Cover with Gram...

The microscope used in the laboratory is what type of microscope? - Compound Binocular Light The ability of a microscope to reveal fine detail is known as? - resolution The 10X eyepiece used in combination with a 45X objective produces a magnification of? - 450X Another name for the 100X objective lens? - oil immersion when putting the microscope away, which objective should be in place? - low power What part of the microscope regulates the amount of light entering? - condenser what part o...

Wet mount steps: - 2. specimen 3. Cover slip over specimen hanging drop steps: - 1. Vaseline around cover slip 2. micro organism in center of cover slip 3. place concave slide on cover slip Gram stain steps: - 1. Crystal Violet 2. Gram's Iodine 3. Ethyl Alcohol 4. Safranin Red negative stain steps: - 1. nigrosin loop ria 4. slide to spread mixture 5. air dry acid-fast stain steps: - 1. carbolfuchsin 2. acid alcohol 3. methylene blue 4. wash & dry simple stain steps - 1. f...

Oxidase Test - *Positive Test:* -Turns blue within 20-30 seconds *Negative Test* -No color change within thirty seconds *Reagent used:* -Oxidase What is the purpose of the oxidase test? - -The tests is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase as an electron acceptor in the ETC. How is the oxidase test performed? - 1. Transfer bacterium to the paper 2. Add drops of complex A & B to bacterium 3. Observe for color change within 30 seconds Why shoul...

How is total magnification calculated? - Multiply the ocular lens (10x) by the objective lens 10x*10=100x 40x*10=400x 100x*10=1000x OCULAR TIME OBJECTIVE what is the function of immersion oil when using the oil immersion objective? - the immersion oil allows for magnification and increases the contrast of the specimen What is meant by the term parfocal? - Once the lens is focused on a particular area it will remain focused on the particular area regardless of changing the objective why ...

What bacterial shape is observed? - Bacillus What bacterial shape is observed? - Coccus What bacterial shape is observed? - Spiral/spirochete How does increase in magnification affect field of vision? - It makes it smaller How does increased magnification affect depth of field? - Depth of field becomes larger Why is it desirable that microscope objectives be parfocal? - So each objective does not require further focusing Which objective focuses the closest to the slide when it is in focus...

Illuminator - Filter for shorter λ Condenser microscope - Focuses light into specimen Diaphragm (iris) - Controls amount of light to specimen Objective lenses at nosepiece - Magnifies image 10, 40, or 100 times Ocular lens - magnifies image 10X Course adjustment - focuses specimen in a broad way Fine adjustment - fine focuses specimen Stage - a platform used to hold the slide containing the specimen Arm - serves as a handle for carrying microscope Mechanical stage adjuster - moves stag...

Primary containment - protection of the lab personnel and the lab environment Secondary Containment - protection of the outside lab environment from exposure to infectious organisms Disposal of biohazards - Place biohazard material in marked bag or container; must be tagged or color coded Disposal of Glass waste - can be washed and reused after autoclaving (Kills station) Disposal of plastic waste - Melt in autoclave to dispose after decontaminated Office hours and location - Zurn 207 Mon...

Simple Stain - Negative Stain - Acid-Fast Stain - Capsule Stain - Endospore Stain - Wet Mount and Hanging Drop - allow observation of living cells to determine motility Flagella Stain - (prepared stain) allows for observation of flagella Columbia CNA with 5% Sheep Blood Agar - differential/selective medium that allows for growth of Gram-positive and inhibit growth of Gram-negative organisms Know the Microscopes Parts! - Base - supports the microscope Mechanical Stage - Condens...

Staph epidermidis - This is MSA and the side labelled "2" is red. Which Staph does this indicate? Staph aureus - This is MSA and the side labelled "1" has turned yellow. Which Staph does this indicate? Lactose Negative - This is an EMB plate. What does the test result of "1" indicate? Lactose Positive - This is an EMB plate. What does the test result of "2" indicate? E. Coli - This is an EMB plate. What is indicated by "3?" Starch was broken down - This is a s...

Compound microscope - 1. Ocular-eyepiece lens (usually 10x) 2. Objective-nosepiece lenses (commonly 4x, 10x, 45x, and 100x) At what magnification do you use the oil immersion technique? - 100x. Total Magnification = - Magnification (Objective) x Magnification (Ocular) Important Parts of the Microscope - Framework, Stage, and Lens System Framework - Arm and base are the structural parts of the microscope which support the basic frame. Stage - Holds the slide. The mechanical stage clamps the...

According to the lab standard operating procedures, you must wash your hands with soap and water for at least _________ before leaving the lab. A) 2 seconds B) 5 seconds C) 15 seconds D) 30 seconds E) 1 minute - C) 15 seconds Which of the following statements does NOT correctly describe a rule that you must follow in the microbiology lab? A) There is no eating or drinking allowed in the microbiology lab. B) If you spill a bacterial culture on your lab coat, you must report it t...

All UTA microbiology labs are considered what biosafety level? - BSL-2 What are four things that all BSL-2 labs must include? - 1. PPE 2. Sinks and eye wash stations 3. Restricted access 4. autoclaves (or alternative decontamination methods) What is the plural for media? - medium media can come in a wide variety of formulations and can exist in what three states? - solid, semisolid, and liquid Catalase is found in both - prokaryotes and eukaryotes What is often considered as a virulence ...

Which staining technique is used to determine the thickness of the peptidoglycan layer based on the cell wall composition? - Gram stain Where would you properly dispose of the following items? - Contaminated slides: Slide trays on student benches Pipettes: Nalgene buckets on student benches Used test tubes: Trash cart at front of lab Uncontaminated broken glass: Broken glass box at front of lab (and notify the TA!) When are open toed-shoes and shorts allowed in the microbiology labs? - Ne...

Give the steps of a PCR - Heat reaction to 95 C to denature DNA Cool rxn to 55 C to allow primers to anneal Heat rxn to 72 C to allow Taq polymerase to bind What machine is used to carry out a PCR reaction? - thermocycler How many cycles does a PCR rxn usually go? - 30 Name the 9 organisms used in the unknown lab - Bacillus subtillis Enterobacter aerogenes Entero cloacae Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus Staphy...

Microscope- The Condenser - focuses the light ging into the objective lens Microscope- The Iris Diaphragm - adjusts the amount of light going into the objective lens Microscope- Nosepiece - holds the objective lenses and rotates to change from one objective to another Microscope- Coarse Objective Knob - moves the stage larger distances to help get the object in focus Microscope- Fine Adjustment Knob - moves the stage smaler increments to help get the object in better focus Microscope- Obj...

What color do Gram-Positive bacteria stain? - Purple What color do Gram-Negative bacteria stain? - Pink What are the 4 steps of a gram reaction? - Primary stain crystal violet, mordant iodine, decolorizer alcohol-acetone, and counterstain safranin. What are some ways you could mess up a gram stain? And how will you know you messed up? - a. Not heat fixing your slide - clear slide, b. over decolorizing - slide has both pink and purple even though the culture was pure, so gram positive bact...

Considering the cultures used to inoculate each medium in this exercise, how many different microbial types should you expect to see in/on each medium? (1.4) - You should only have 1 if using good sterile technique. A successful aseptic transfer will have only micrococcus lutes growth- this would be a pure culture You were asked to describe differences in appearance of growth in each culture, if present. In which medium was this the most difficult to determine? What made it difficult? (1.4)...

Proper use of the microscope - all specimens must be viewed under cover glass, focus with scanning objectives first, use course adjustment knob, once in focus, use the fine focusing knob (when viewing with low or high power objectives) Depth of Field - refers to the thickness of the plane of focus Field of View - area that is visible when using the microscope Why use a microscope? - To view and study the microscopic world of organisms and objects less than 5mm in size that cohabitate with ...

gram-positive - appear purple gram-negative - appear pink Isolation by Dilution - -reducing number of bacteria so that individual colonies may be isolated -streak plate technique is commonly used to separate mixed cultures to isolate colonies that can be used to make a pure culture Selective media - promotes the growth of some organisms and inhibits the growth of others Differential media - show different growth characteristics for certain organisms, such as a color change TSA - Tryptic ...