Micro lab midterm study guide PDF

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Microbiology Lab 3051 (Spring 2020) Mid-Term Laboratory Exam Study Guide 1. Know the parts of the microscope and their functions.

2. Know how to use the microscope (adjusting for proper viewing, getting an image into focus, using the oil immersion lens) and how to take care of a microscope. 3. Describe aseptic technique and understand why it is important. 1. 2. 3. 4. 5. 6.   

Flaming the loop sterilizes it tube cap is removed flaming the tube tip sterilizes the surface only sterilized portion of loop enters tube the tube is flamed, tube is recapped loop is flamed again. Allow the loop to completely cool before obtaining your culture. Aseptic technique prevents contamination of culture from foreign bacteria in the environment. Also protect against microbes being released into environment and infecting people

7. Define and describe culture media. 

Culture media- nutrient preparation devised to support the growth (reproduction) of microorganisms o Includes macronutrients, micronutrients, trace metals, and growth factors o Agar plate, agar slant, nutrient broth, agar deep tube

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Defined media- exact composition (quantitative and qualitative) is known Complex media- contain some ingredients unknown composition and/or concentration o Common complex media components  peptones, extracts, agar General purpose media- support the growth of many microorganisms (TSA) Enriched media- general purpose media supplemented with highly nutritious substances such as blood (blood and chocolate agar) Minimal media- contains the minimal necessities for growth of the wild type o Only contains inorganic salts, a simple carbon source, and water Selective media- favor the growth of some microorganisms and inhibit the growths of other (EMB agar) Differential media- distinguishes between different groups of microorganisms based on their biological characteristics (blood agar and MacConkey)

 8. Compare and contrast how to make a bacterial smear from liquid media and solid media. 

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When preparing a smear for a solid media you first add a loop full of water to the slide, then obtain your solid media with a sterilized and add it to the water on the slide. Allow it to air dry and then either heat or chemically fix the slide. When preparing a smear for liquid media, use a sterilized loop and aseptic technique to obtain a loopful of liquid media and add it to the slide. Allow it to air dry and heat/chemically fix the slide

9. Explain the importance of heat fixing and air-drying specimens on slides.     

heat fixing causes the cells to adhere to the slide so that the application of dye or water doesn’t washes them off it kills the microorganisms allows the microorganism to be stained better by causing pores in the cell wall to open some bacteria cannot withstand high temperatures because they are too fragile o use chemical fixing when media is delicate (ex. Flagella or endospores) air drying prevents any aerosols from being released into the environment when heated

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Compare and contrast simple staining and differential staining.

Simple staining  use of a single stain o For visualization of morphological shape and arrangement

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Differential staining  use of two contrasting stains separated by a decolorizing agent o Identification and visualization of structure o Divides microorganisms into groups based on their staining properties: gram stain, acid fast stain, endospore stain, capsule stain

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Recognize the different bacterial morphologies.

o Staphylococci  clusters o Tetrads  4 cocci in a square o Sarcinae  cubic configuration of 8 cocci Bacilli  rods o Coccobacilli  very short rods Vibrios  resemble rods, comma shapes Spirilla  rigid helices Spirochetes  flexible helices Mycelium  network of long, multicellular filaments Pleomorphic  variable in shape Palisades  picket fence, stuck together from tip to end

12. Compare and contrast gram-positive and gram-negative cells (what colors they stain using the Gram stain and how cell wall structure determines how they stain).  

Gram positive cells stain purple and have a thick peptidoglycan layer in their cell wall Gram negative cells stain pink and have a thin peptidoglycan layer in

13. Know all of the steps and stains used in a Gram stain. Know the purpose of each stain and how bacteria look at each step. Know which stain is the primary stain, counterstain, mordant, and decolorizing agent. 1. Apply crystal violet as the primary stain to a heat fixed slide, let sit for one minute, and rinse with water a. Both gram negative and gram positive cells will appear purple at this stage 2. Apply gram iodine as the mordant, let sit for one minute, and rinse with water a. both gram negative and gram positive cells will appear purple at this stage 3. apply alcohol to decolorize the cells, let sit for 20 seconds, and rinse with water a. at this stage, gram positive cells will be purple and gram negative cells will be colorless 4. lastly apply safranin as the counterstain, let sit for one minute, and rinse with water a. at this step, gram positive cells will appear purple and gram negative cells will appear pink 14. Understand the purpose of a streak plate and how you would do one.  

Streak plating is used to obtain a pure culture and isolated colonies Pure culture  population of cells arising from a single cell o Allows for the study of single types of microorganisms o A mixture of cells is applied to an agar surface so that individual cells are well separated from each other

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Define pure culture and CFU.

Pure culture  population of cells arising from a single cell CFU colony forming unit, is a unit used to estimate the number of viable bacteria or fungal cells in a sample

16. Describe the growth of bacteria in various types of media (cultural characteristics) using the proper microbiological terms.

17. Know the purpose of a gelatin stab, how you conducted this test, and what a positive reaction for gelatinase production (gelatin hydrolysis) would look like.     

Gelatin hydrolysis test is used to determine the ability of an organism to produce extracellular proteolytic enzymes, gelatinases that hydrolyze gelatin. Gelatinases hydrolyze gelatin into polypeptides and then polypeptides are further converted into amino acids To inoculate the gelatin media use a sterilized needle that has been dipped in culture and stab the gelatin cleanly and deeply and immediately remove Positive result  gelatinase enzymes are present in the organism and is able to liquify the gelatin media Negative results  gelatinase enzymes are not present and the media is not liquified

18. Describe endospores, their function, and know which genera produce them.

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Endospores  complex, tough, non-reproductive, dormant survival structures formed by some bacteria from the firmicute family Resistant to heat, radiation, chemicals, and desiccation The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress Produces by the following genera  bacillus, clostridium, clostridioides

19. Understand the Schaeffer-Fulton endospore staining procedure (know the primary stain and counterstain), why heat is used as a mordant, and what endospores and their producers look like under the microscope after being stained. Endospore staining procedure Set up a hot water bath and bring to a boil Place a small paper tower over the heat fixed slide Saturate the paper towel with malachite green (the primary stain) and place over the hot water bath and allow to sit for 5 minutes/rinse with water 4. Cover the smear with safranin (counterstain) for 30 seconds and rinse with water/blot slide with paper towel  Heat is used as the mordant because it drives the stain deep into the endospore. Endospore are very tough and resistant and would not be able to be stained without a mordant  The vegetative cells appear pink  The endospores will appear green  1. 2. 3.

20. Know what genera of bacteria are acid-fast and what characteristic causes them to be acid-fast.  

Acid fast staining is useful for the genera mycobacterium High lipid (mycolic acid) content in cell walls is responsible for the acid fast staining characteristics

21. Describe the acid-fast stain (primary stain, decolorization, counterstain) and know how acid-fast cells and non-acid fast cells appear after staining.  Acid fast staining procedure 1. Cover a slide with carbolfuchsin (primary stain) for 5 minutes and rinse with water 2. Dip the slide in acid alcohol (decolorizer) until the runoff is clear and rinse with water 3. Cover the slide with methylene blue (counterstain) for one minute and rinse with water/blot slide with a paper towel  Acid fast bacteria will appear red  Non acid fast bacteria appear blue

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Describe bacterial capsules and their functions.

Capsules are usually composed of polysaccharides (sugar) and are well organized and not easily removed from cell Advantages of capsule layers include: o Attachment to surfaces and each other (biofilms) o Protect against desiccation, phagocytosis, viruses, detergents, toxins, antimicrobials, and absorption of nutrients The capsule is considered a virulence factor because it enhances the ability of bacteria to cause disease

23. Describe the use of negative staining to visualize capsules and know how capsules appear after using this method.   

Negative staining  capsules appear colorless against a stained background Negative staining is used with capsules because their sugar layer prevents the stain from adhering to their cell Used to study the morphological shape, size and arrangement of bacteria that are too difficult or too delicate to directly stain

24. Understand the method we used to visualize capsules, including the purpose of Maneval’s A and Maneval’s B.  1. 2. 3. 4. 5. 6.  

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Capsule staining procedure (negative staining) Place Maneval’s A near one end of a flamed slide Place a small amount of culture within the Maneval’s A Using a clean slide, spread the stain across the entire flamed slide After the smear air dries, heat fix the slide Flood slide with Maneval’s B and let sit for one minute Drain the stain off the slide, but do not rinse with water. Let air dry and observe Maneval’s A (Congo red) has a pH indicator that turns blue following the addition of Maneval’s B Mavenal’s solution is a combination of acetic acid and acid fuchsin. The acetic acid lowers the pH in the sample and causes the congo red to change from red to blue. The acid fuchsin penetrates through the capsule and stains the cell a bright red Will appear to have a dark red background and the capsules will remain colorless

25. Understand the methods we used to see motility and how motility (or lack of) was determined.  Hanging drop procedure (this is the test for motility) 1. With a toothpick, dot vaseline on the four corners of a cover slip

2. Place a drop of culture in the center of the cover slip 3. Lower a depression slide, concavity facing down, over the drop on the cover slip and press to seal the Vaseline 4. flip the depression over and observe with microscope on 40x and look for movement  a soft agar stab inoculation can also be used to determine motility of an organism o if the culture spreads throughout the agar after incubation, then motility is present. If there is no growth, then motility is not present 26. Of the bacteria we tested for motility, be able to name which bacterium was motile and which one was non-motile. 

E. coli was motile but M. luteus is not motile

27. For each of the following tests, know the purpose, what a positive reaction would look like, what media and/ or reagents were used, and how the media and/or reagents work to show negative and positive reactions (ex. methyl red in MRVP, turns red when the pH drops to 5 or less indicating mixed acid fermentation).

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Carbohydrate (Glucose) Fermentation Purpose  carbohydrate fermentation test is used to determine whether or not a bacterium can utilize a certain carbohydrate. It tests for the presence of acid or gas produced from carbohydrate fermentation. Positive result  the development of a yellow color in the medium. Gas formation is indicated by the appearance of gas bubbles in the Durham tube Negative result  lack of yellow color development Media/reagent used  carbohydrate broth (glucose) with Durham tube How media/reagent works to show positive/negative result  carbohydrate fermentation is the process microorganisms use to produce energy. The media in each tube contains a single carbohydrate- glucose, lactose, and sucrose. The bacteria used for inoculation will ferment these if they are capable and produce a gas as a biproduct. Methyl Red Purpose  detects the production of sufficient acid during the fermentation of glucose. Used to determine whether or not the unknown bacteria can produce large quantities of stable acids. It detects organisms capable of doing mixed acid fermentation Positive result  methyl red is added to the MR-VP broth and the color will be red if positive

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Negative result  turns yellow after addition of methyl red reagent to the MR-VP broth Media/reagent used  MR-VP broth and methyl red How media/reagent works to show positive/negative result  MR-VP is a simple broth that contains peptone, buffers, and dextrose or glucose. Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. Some of these pathways produce unstable acidic products. Methyl red is a pH indicator which is red in acidic solutions and yellow in basic solutions Voges-Proskauer Purpose  used to determine if an organism produces acetylmethyl carbinol from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in the presence of naphthol, strong alkali, and atmospheric oxygen. Used to detect acetoin in a bacterial broth culture Positive result  cherry red color means that acetoin is present Negative result  yellow brown color indicates that acetoin is not present Media/reagent used  MR-VP and Barritt’s A reagent and Barritt’s B reagent Citrate utilization Test Purpose  used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic ammonium dihydrogen phosphate is the sole fixed nitrogen source Positive result  blue slant and uses citrate as carbon source Negative result  green slant and no growth and does not use citrate as carbon source Media/reagent used  citrate agar slant (stab and then streak) Nitrate Reduction Purpose  nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. Also test the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. Positive result  no color change Negative result  color change to red (nitrate was present and reduced to nitrite) Media/reagent used  nitrate reduction broth with Durham tube/solution A/solution B/zinc dust How media/reagent works to show positive/negative result  nitrate broth contains nutrients and potassium nitrate as a source of nitrate. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid Oxidase Test

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Purpose  oxidase test is used to identify bacteria that produce cytochrome c. oxidase, an enzyme of the bacterial electron transport chain. All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration Positive result  positive when the color changes to dark purple within 5 to 10 seconds Negative result  negative when no color change Media/reagent used  TSA place Catalase test Purpose  the enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs Positive result  bubbles are a positive result for the presence of catalase Negative result  no bubbling Media/reagent used  nutrient agar slant/hydrogen peroxide Starch Hydrolysis Purpose  purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth. Use of starch is accomplished by an enzyme called alpha amylase Positive result  clearing zone indicated the organism has hydrolyzed starch Negative result  blue, purple, or black coloration of the medium Media/reagent used  starch plate/gram’s iodine Casein Hydrolysis Purpose  to test for the digestion of casein by proteolytic exoenzymes that are secreted outside the cell Positive result  clearing zone observed around colony growth indicates hydrolysis Negative result  no clearing zone Media/reagent used  skim milk plate How media/reagent works to show positive/negative result  casein hydrolysis is tested by growing an organism on skim milk agar plate and then checking the plates for hydrolysis. H2S Production Purpose  this test determines whether the microbe reduces sulfurcontaining compounds to sulfides during the process of metabolism Positive result  black color produced Negative result  no back color produced Media/reagent used  SIM

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How media/reagent works to show positive/negative result  if sulfide is produces, it combines with iron compounds to produce FeS, a black precipitate Indole Production Purpose  used to determine the ability of an organism to split amino acid tryptophan to form the compound indole. Tryptophan is hydrolysed by tryptophanase to produce three possible end productsone of which is indole Positive result  pink layer on top of media Negative result  yellow layer on top of media Media/reagent used  SIM tube/kovac’s reagent Urease Test Purpose  used to differentiate organisms based on their ability to hydrolyze urea with the enzymes urease Positive result  bright pink Negative result  orange Media used  urea slant

28. Know the functional type, mechanism of action, what type of colonies will grow, and how they can be distinguished on the following media: MacConkey Agar (MAC), Phenylethyl Alcohol Agar (PEA), and Blood Agar. 

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MAC o Functional type: selective and differential o Mechanism of action: salts inhibit gram positive growth, lactose and pH indicator (neutral red) detect lactose degradation o Colonies: gram negative; lactose fermenters are pink, non lactose fermenters are white/colorless PEA o Functional type: selective o Mechanism of action: phenylethyl alcohol inhibits gram negative o Colonies: gram positive Blood agar o Functional type: differential and enriched o Mechanism of action: some cells degrade hemoglobin, some do not o Colonies: both; gamma agar around colony remains unchanged, alpha agar under colony is dark/greenish, beta agar and colonies are small, opaque or translucent (slightly yellowish)

29. Understand how to construct a bacterial growth curve by measuring the turbidity of a broth culture.

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Time is the x-axis, absorbance is y-axis Use spectrophotometric measurements every 20 minutes then plot

30. Know and describe the four phases of bacterial growth in a batch culture. 1. Lag  bacteria adapt themselves to growth conditions. It is the period ...