Microbiology Lab Study Guide (2) PDF

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Microscopy: Use and Care Microscope: an instrument for viewing objects that are too small to be seen by the naked eye We use: “Brightfield Compound Light Microscope” Advantage: provides good contrast for stained specimens Magnifies object using light and lenses Compound: passes light through specimen and then through two different lenses PARTS OF MICROSCOPE! Ocular lens: have a magnification of 10x Objective lens: Scanning (10x): shortest lens used to scan whole slide Low Power (20x): longer than scanning lens, and used to view objects in greater detail High Power (40x): to view object in even greater detail Oil immersion (100x): used with immersion oil to view objects with greatest magnification Why use oil: if no oil the refraction happens: light bends as it passses through glass Coarse adjustment knobs: To raise and lower the stage To bring close or further from objective lens Fine adjustment knobs: 2 knobs in the center of each coarse adjustment knob Used for precision focusing How to focus the microscope: -Place slide on stage -Start with 10x lens and find the object/specimen by moving coarse adjustment knobs -Once the specimen is found, the microscope is focused -Now, we will move to the 20x lens, by rotating nosepiece and again focusing the specimen by slowly moving fine adjustment knob (you don't have to use coarse adjustment knob after 10x) -After that, we will see our specimen at 100x by using oil immersion lens Total magnification= magnification of objective lens x magnification of ocular lens What is the total magnification used to view bacterial cells through the ocular lens of 4x and objective lens of 100? 4x (ocular) x 100x (oil immersion objective) = 400 Fine adjustment knob (40x or 100x) Coarse adjustment knob (10x or 20x) At end always place the scanning lens (10x) into position Field of view: Amount of specimen that is visible It will decrease with increase of magnification

Aseptic Technique: Transfer of bacteria/ Isolation of bacteria Aseptic Technique Subculturing: inoculating, process of taking a colony from a mixed culture Microbes are always referred to by their scientific name: Genus name- always capitalized Species name- lower case Italicized or underlined In order to grow a bacteria you need to know: Nutritional requirements: energy source, CO2, N2, mineral, and growth factors Temperature: approximately 37 degrees celsius (most bacteria) O2 requirement: Strict/ obligate aerobe: O2 necessary for survival (grow on top of tube) Strict/ obligate anaerobe: die in presence of O2 (grow on bottom of tube) Facultative anaerobe- can survive with or without O2 (grow throughout tube) Microaerophilic- requires reduced levels of O2 gas and higher levels of CO2 gas Transfer bacteria: Culture medium: a nutrient material prepared for the growth of microbes in a lab Broth: Liquid medium Used to grow large quantities of bacteria in a small space Inoculation using inoculating loop Semi-solid Deep: Semi-solid medium (0.5%- 0.7% agar) Test for O2 requirements or motility Inoculated by inoculating needle Slant: Solid medium For long-term storage and easy transport of microbes Inoculate using inoculating loop Plate: Solid medium (1.5% agar) For isolation of bacteria and to obtain isolated colonies Inoculation by inoculating loop Inoculate- to introduce microbes to a media -inoculating loop and inoculation needle Agar: An extract derived from a marine algae is called agar It makes the culture media solid Aseptic technique: is a series of steps used in lab to prevent contamination Incubation: once you have inoculated your culture media you want to incubate it so that your microbes will grow

Incubation involves placing your microbe in an environment that provides the correct optimal temperature Result: what we can see with our eye Broth: 1) Growth at bottom of tube: sediment 2) Cloudy broth: turbidity 3) Film of membrane floating on top of broth: pellicle 4) Clumps of bacterial floating on top of broth: flocculant Semi-solid Deep: 1) Growth at top of deep: strict/ obligate aerobe 2) Growth from top to bottom of deep: facultative anaerobe 3) Growth only at bottom: strict anaerobe 4) Growth that spreads away form inclutation point or “inverted” christmas tree: motile organism 5) Growth only along inoculation point: non-motile organism Slant: 1) Branched growth: arborescent 2) Small colonies: beaded 3) Root-like growth: rhizoid 4) Pointed growth: echinulate 5) Growth that grows beyond the inoculation point: effuse 6) Even growth along inoculation point: filiform Plate: 1) Whole colony appearance a) Circular, irregular, biconvex, filamentous, rhizoid, spindle 2) Margin/ edge a) Entire, undulated, lobate, filamentous, serrate, scalloped 3) Elevation a) Flat, raised, convex, pulvinate, umbonate 4) Size a) Punctiform, small, moderate, large Interpretation: what it means All plates must be inverted during inoculation to prevent condensation from falling on the surface of the agar and distorting the growth on the plate Isolation of bacteria: 3 dilution techniques: Pour plate: only one that uses empty sterile petri plate, then bacteria grows in AND on agar (bacteria mixed with agar and poured) Spread plate: spread on solid medium in a petri plate (spreading rod)

Streak plate: streak on solid medium in petri plate (inoculating loop) Purpose: to dilute the original concentration of microbes so that individual isolated colonies form Colony: a viable mass of genetically identical (clone) microorganisms all originating from a single mother cell Steak plate method: 4 quadrant-- dilute bacteria with each quadrant Mixed , contaminated, and pure culture= results Mixed culture- more than one microbe made up the original bacterial culture Isolate colonies must be present on a plate in order to determine whether the bacterial culture is a mixed color- different colors Contaminated culture- unwanted microbes are present in the original bacterial culture Isolated colonies must be present on a plate in order to determine whereter the bacterial culture is a contaminated culture - organisms look different Pure culture- only one microbe was present in the original bacterial culture Isolate colonies must be present on a plate in order to betere whether the bacterial culture is a pure culture- all same Isolating a specimen in pure culture is the first step towards identification of unknown microorganism Mixed culture are not useful because inability to determine which organism is responsible for any observed results

How to Prepare a Smear and Direct Staining: Methylene Blue/ Negative Staining How to prepare a smear and direct staining: methylene blue: Most microorganisms appear colorless when viewed through a microscope Staining is used to observes the structure of microbes under microscope Advantages of staining techniques: Increases contrast of the specimen Allows us to determine: Morphology: shape of cells Bacilli- rod like shape Cocci- spherical shape Spirilla- spiral like shape Size Arrangement: how cells are positioned in relation to one another Single:one Diplo: paried Strepto:chains Staphylo:grape cluster Sarcinae: 3D cubical structure

Tetrade: cubical Further characteristic of bacteria Staining bacteria: Stain: a dye that binds to a particular cellular structure and allows the bacteria to appear Two different types: Simple stains Consists of a single dye Determines shape, size, and arrangement of cells Difference between components cannot be determined Common stains: methylene blue, safarin, crystal violet Direct Staining Only stains the bacteria Background remains colorless Bacteria- negatively charged and dye is positive so they attract and make the bacteria visibleMethylene blue dye (basic die) Smear: thin film of cells that have been placed on a slide Heat fix slide: briefly pass through burner flame 2 to 3 times Kills bacteria, causing them to adhere to a slide Causes changes in cells that enable them to take up stains more readily Negative Staining Differential stains Composed of 2 or more dyes Can differentiate between organisms cellular components Common type: gram staining Chromophore: the ion that gives a stain its pigment and its charge Acidic dyes: negative chromophore Basic dyes: positive chromophore Negative Staining: A technique that does not stain bacteria but stains the background Bacteria will appear clear against stained background Useful when morphology or size is difficult to determine Type of staining reagents used: nigrosin (acidic dye- negative charge) Negative dye + negative bacteria repel each other = does not stain- is clear Heat fixation is not used Gram Staining

Simple staining vs Differential staining Simple: one dye, difference between cell components cannot be determined, common stains: methylene blue, safarrin, and crystal violet Differential: 2 or more dyes, difference between cell components can be determined common stains: gram staining Gram staining: Differential staining technique: allows to classify whether the bacteria is gram negative or gram positive Basic steps: 1) Crystal violet/ primary stain a) Crystal violet is a basic stain, meaning it has positively charged chromophore b) Stains bacteria purple 2) iodine/ mordant (dye-fixing agent) a) The iodine binds with the crystal violet cell to form the CV-I complex i) (crystal violet- iodine complex) 3) ethanol/ decolorizing agent a) Allows primary stain to be washed out of some bacterial cells, while other bacterial cells are not affected (negative- turn white) 4) safranin/ counterstain a) Basic stain (positively charged chromophore) b) Stians the bacteria that have been decolorized by ethanol (negative) and stains them red -gram positive- purple result -gram negative- red result Gram reaction: whether bacteria is gram negative or gram positive Positive- thick peptidoglycan layer Negative- smaller peptidoglycan layer, lipopolysaccharide layer (washes away) and has periplasmic space Be careful with the amount of ethanol you are providing- too much or too little = bad Under and over decolorization: Under- Less ethanol: Positive: does not affect the cell- stays purple Negative: layer does not break down- stays purple (false result-false negative) Over- More ethanol: Positive: peptidoglycan layer breaks down- stains red (false result-false positive) Negative: does not affect cell- stains red Procedure: Prepare smear

For solid culture: a small portion of the solid culture is mixed with water and spread over the surface of a glass slide and air-dried Air-dry semar and heatfix

Introduction to Special Media/ Carbohydrate Catabolism Introduction to Special Media: Biochemical Test: Allows us to determine a microorganism metabolism Metabolism- chemical reactions occurring within all living organism Involves the use of endoenzymes and exoenzymes Endoenzymes: produced inside of cells and function inside cells- further break down of small molecules Exoenzymes: produced inside cell but function outside the cell- breakdown larger molecules that cannot fit inside the cell and then those are broke down in the cell by endoenzymes Basic metabolic mechanisms: Simple sugar (glucose)-- (endoenzyme)--- acid Complex sugar (sucrose/lactose)--(exoenzyme)--simple sugar---(endoenzyme)--acid Amino acid (tryptophan)---(endoenzyme)---ammonia Protein (peptone)--(exoenzyme)--amino acid(ornithine)---(endoenzyme)--ammonia Specialized Media: Differential media: allow to distinguish one bacterium from another by how they metabolize or change the media with a waste product- change in color..etc Selective media: designed to enhance growth of some organisms by inhibiting other organism Enrichment media: enhance growth of desired bacteria but other bacteria will still grow as well Eosin Methylene Blue (EMB) (selective for gram negative bacteria) Contains: eosin, methylene blue, agar, lactose, peptone Eosin and methylene blue: inhibit growth of Gram positive, but permits growth of gram negative bacteria (dyes) If a gram negative bacteria is able to ferment lactose, the eosin dye will react to the acid and turn the bacterial growth pink, purple, black or metallic green-- positive result -thick and strong growth = gram negative -color change= positive for lactose fermentation -what is the primary source of growth of A and B bacteria- EMB bacteria: -peptone (look at ingredients) or lactose Phenylethyl Alcohol (PEA) (selective for gram positive bacteria)

Contains: Phenylethyl alcohol, peptone (protein source), and agar (solidifying agent) Phenylethyl alcohol peptone inhibits growth of Gram negative organism but interfering with their DNA synthesis. Selective for gram positive bacteria -thick/ opaque growth- gram positive After streaking, the plates are incubated in the inverted position at 37 degrees C Carbohydrate Catabolism Catabolism is the breakdown of larger molecules into smaller molecules Oxidative catabolism- aerobic condition; oxygen also functions as final electron receptor Fermentative catabolism- facultative anaerobic and strict aerobic condition Does not require oxygen to breakdown molecules but can occur in the presence of oxygen Starch Hydrolysis Test Contains: starch, beef extract (peptone-protein), and agar It is too large, in its original form, to pass through the bacterial cell membrane Only those organisms that are capable of producing exoenzymes can break down starch Starch-- (exoenzyme-amylase)---glucose---(endoenzyme)---acid Iodine will detect the presence of absence of starch in the vicinity around the bacterial growth Iodine reacts with starch and produces a blue, black, or dark brown color (negative result becauses scratch has not been hydrolyzed) Therefore, starch hydrolysis is a clear zone surrounding the bacterial growth -clearing of plate after addition of iodine= + for amylase and scratch hydrolysis Oxidation- Fermentation Glucose (OF Glucose) Contains: peptone, a small amount of agar (for determination of motility), pH indicator(bromothymol blue) and glucose Bromothymol blue Yellow - acid produced Green - neutral pH Blue- basic products -This test will be conducted using two OF-deeps -Both deeps will be inoculated with needle -One deep- covered with mineral oil- to create anaerobic environment -Other deep- left as it is- to create aerobic environment -Strict aerobes that are capable of utilizing glucose, will only be able to oxidize glucose in “aerobic” tube. Results into yellow color, which means acid in the medium- oxidation has occurred and acid has been produced -“Anaerobic” or “sealed” tube will remain green - no fermentation process -Facultative anaerobes that are able to utilize glucose will turn “aerobic” tube and

“anaerobic” tube medial yellow (oxidation has occurred and acid has been produced) This will indicate that that organism can ferment glucose (anaerobic tube turns yellow) Fermentation does not require oxygen, but can take place in the presence of oxygen Can also read for: oxygen requirements and motility -yellow-facultative anaerobe -green and yellow-strict aerobe -green-control -lighter green, one has a little blue on top- strict aerobe Fermentation of Carbohydrates/ Respiration Fermentation of carbohydrates True fermentation: production of both acid and gas Fermentation: production of acid only Peptone utilization: utilization of peptone instead of sugar: basic end product Carbohydrate Fermentation Test Contains: peptone, carbohydrate, durham tube Phenol red (pH indicator) Red: neutral Yellow: acidic Fuchsia pink: basic Basic Metabolism Mechanisms: Fermentation/ True Fermentation Simple sugar (glucose)---(endoenzyme)--acid Complex sugar(sucrose)---(exoenzyme)---simple sugar---(endoenzyme)--acid Peptone Utilization Amino acid (tryptophan)--(endoenzyme)--ammonia Protein (Peptone)--(exoenzyme) amino acid(ornithine)--(endoenzyme)--ammonia End products: When gas is produced, trapped in the inverted Durham tube - appears as a bubble -yellow color- + fermentation -red/ pink- - fermentation, + for peptone utilization -gas bubble- + gas production (true fermentation) If an organism is not capable of fermenting the sugar, then it will utilize peptone and therefore ammonia, turning the color to pink, darker red, or orange. Carbohydrates no more- yellow then red to pink Respiration Oxidation: loss of electrons Reduction: gain of electrons In respiration inorganic molecules serve as the electron acceptor

-aerobic respiration: requires oxygen, the oxygen will be the final electron acceptor -anaerobic respiration: organism cannot survive in the presence of oxygen, meaning that an inorganic molecule other than oxygen acts as the final electron acceptor Oxidase Test: Cytochromes- part of the electron transport system Cytochrome C (cytochrome oxidase) transfer electrons to oxygen thus reducing it to Water Endoenzyme It will change the oxidase strip to a purple color ONLY STRICT AEROBIC organisms produce Cytochrome C-- give positive results -oxidase strip blue, purple, black- + for cytochrome C Catalase Test Catalase breaks down H2O2 (hydrogen peroxide) into water and oxygen Endoenzyme, bacteria that are capable of producing catalase will produce bubbles when mixed with H2O2 All organisms that use O2 produce catalase. Strict aerobic or facultative anaerobic microorganisms produce catalase -bubbles after addition of Hydrogen Peroxide- + for catalyst Nitrate Reduction Test It is an anaerobic process so it uses nitrate instead of oxygen as the terminal electron acceptor NO3 (nitrate)-- NO2 (nitrite)--- N2 (nitrogen) Addition of reagent A and B Nitrate broken into nitrite- reddish color production If not red add zinc-- colorless= + nitrogen gas Color red- zinc acts as reducing agent: nitrate to nitrite-- negative for nitrate reduction because zinc did process instead of organism

Protein Catabolism Proteins are made of amino acids which consist of carbon, hydrogen, nitrogen… Deamination: the process of removing an amine group from an amino acid The amine group is converted into ammonia Decarboxylation: is the removal of carbon dioxide from an amino acid and will change the pH to basic- alkaline Urease Hydrolysis Test Contains: urea, yeast extract, phenol red (in carbohydrate fermentation exercise- pH) Urea + H2O----(urease- exoenzyme)---NH3(Nitrate-alkaline)+ CO2 pH indicator turns fuchsia pink (urease positive organism) -alkaline product Urease negative organisms will turn the medium yellow due to acidic byproducts being produced from the fermentation of yeast extract

-pink- + for urease production and + for urea hydrolysis Gelatinase Hydrolysis Test Contains: beef extract, peptone, gelatin Gelatin is a protein, only organisms that are able to produce exoenzyme- gelatinase can use gelatin When gelatin has been hydrolyzed the medium will turn into a liquid state Those organisms that cannot produce gelatinase will use beef extract and peptone (protein) for survival but the medium will remain solid Liquid- + for gelatinase production and + for gelatin hydrolysis Incubate at room temperature for 4-7 days Motility, Indole, Ornithine Test (MIO) Contains: peptone, glucose, bromocresol purple (pH indicator), Tryptone (provide the amino acid tryptophan), ornithine Decarboxylation of ornithine results in alkaline end products that turn the medium into a darker shade or purple Decarboxylation of Ornithine: Ornithine yellow---(ornithine decarboxylase) Putrescine purple Motility and indole production can also be checked. Tryptophan ----(tryptophanase)--- indole Indole- Kovacs Reagent---red ring -red ring after Kovacs reagent: + indole and tryptophan yellow=negative -growth moves away from inoculation: + for motility -darker purple color change: + ornithine decarboxylation and ornithine decarboxylase No color change or yellow= negative 1) Motility 2) Ornithine decarboxylation 3) Indole production and tryptophanase

______________________________________________________________________________ Bacteria of the Skin: Symbiosis: Interaction between two different organisms living in close physical association typically to the advantage of both Commensalism: An organism benefited, while the other organism is neither benefited or harmed Mutualism: Both individuals benefit Parasitism: One organism benefits at the expense of the other

Skin is an inhospita...