Microbiology Laboratory Exam

Safety and Laboratory Guidelines Infectious agents are categorized into four safety levels: 1. BSL-1: Organisms do not typically cause disease in healthy individuals and present a minimal threat to the environment and lab. 2. BSL-2: Organisms are commonly encountered in the community and present a moderate environmental and health hazard. These organisms are associated with a variety of human diseases, most of which can be successfully treated if identified in a timely manner. 3. BSL-3: Organisms are of local or exotic origin and are associated with respiratory transmission and serious or lethal diseases where treatment and vaccines may not be available. Special ventilation systems are used to prevent aerosol transmission out of the laboratory, and access to lab is restricted. 4. BSL-4: Organisms have great potential for lethal infection. The lab is isolated from other facilities. Student Conduct: - Wash hands thoroughly with soap and water at the beginning and end of lab, also after handling living microbes. - Sanitize lab bench area with dilute bleach solution at beginning and end. Dispose of paper towels in general waste baskets. - If a spill happens, surround the droplets with a circle of bleach and use paper towel to absorb the spill → dispose contaminated paper towels in biohazard bag in morgue. - Do not remove any organisms or chemicals from the laboratory. - Do not perform any transfers over books/papers because you may inadvertently and unknowingly contaminate them with droplets or aerosols that settle. - Never hold a tube culture by its caps because caps are generally loose to allow aeration and are not secure enough to be used as a handle. - Plastic Petri dishes should be labeled on their base, not on their lid, because the lid may get separated from its base during reading or rotated from its correct orientation. - Sterilization of inoculating instruments is done in the hottest part of the Bunsen burner flame - the tip of the inner cone. Heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items may be done in the outer cone. - Carry a microscope with both hands, one grasping the arm and other supporting the base. - When done with the microscope, center and lower the mechanical stage. Lower the light intensity to minimum and turn off the light. Clean any oil off with lens paper. - Lens paper is used for cleaning the condenser and objective lenses. - We do not use gloves because they lessen sensation and may result in unrecognized contamination. Where things go: - Instruments or glassware (ex: small beakers for alcohol) are returned to side counter. - Microscope slides prepared from heat-fixed specimens (ex: Gram stain) should be disposed of in labeled enamel pan on window sill in back of room. - All contaminated tubed media is returned to morgue. - Contaminated Petri dishes are placed in large biohazard bag located on top of the counter in morgue. Never place pointed or tubed media in biohazard bag. Dispose uncontaminated glass in broken glass box at the back of room. Slides used for wet mounts of noninfectious specimens can be washed and reused. Once items are in morgue, they cannot be taken out. Aseptic Transfers and Inoculation Methods Media: - Nutrient Agar: Agar slant (used when we want to grow bacteria over short period of time, can be refrigerated after incubation, and maintained for several weeks), Agar deep (stab needle in to more anaerobic environment inside), or Agar plate (used for obtaining isolation of species). - Nutrient Broth: Used to grow microbes when fresh cultures or large numbers of cells are required. Usually 8-10 mL is broth. Both Agar & Broth contain carbon, phosphorus, sulfur, nitrogen, and other growth factors - Culture = a medium that contains living microbes Pure culture = a culture that contains a single species - Mixing broth by hand: A broth culture should always be mixed prior to transfer by tapping the tube with fingers Holding tubes at angles: tube is held at an angle to minimize chance that airborne microbes will drop into it Move the tube not the loop. Keep the loop hand still and don’t catch the loop on the tube’s lip when removing it. Fishtail inoculation of an agar slant: begin at the base of the slant and gently move the loop back and forth as you withdraw the tube. Be careful not to cut the agar. - Inoculating Instruments (from left to right): - Serological pipette - Disposable transfer pipette - Pasteur pipette - Inoculating needle - Inoculating loop - Disposable inoculating needle/loop - Cotton swab - Glass spreading rod (not an inoculation instrument, but used to spread an inoculum introduced tro agar plate by another instrument) Microscopy and Staining Ocular Lens Objective Lens Total Magnification Scanning 10x 10x 100x Low Power 10x 20x 200x High Dry 10x 40x 400x Oil Immersion 10x 100x 1000x Total Magnification = Magnification by Objective Lens x Magnification by Ocular Lens Objective (with 10x oculars) Field Size 10x 2000µm 20x 1000µm 40x 500µm 100x 200µm - - - - Always start at lowest power Condenser = second set of lenses that don’t magnify, but concentrate the light and make illumination of the specimen more uniform Iris diaphragm = controls how much light coming from condenser When adjusting microscope, 1st adjust condenser then 2nd iris diaphragm Working distance = distance between lens and slide → decreases as magnification increases Numerical aperture = the measure of a lens’s ability to capture light coming from the specimen and use it to make the image Because most biological specimens are transparent, the contrast between the specimen and the background can be improved with stains to the specimen. However, keep in mind that staining process usually kills cells The best limit of resolution achieved by a light microscope is about ~0.2µm Bright field microscopy = the specimen restricts light transmission and appears “shadowy” against a bright background Dark field microscopy = a special condenser is used so only the light reflected off the specimen enters the objective. A brightly lit specimen appears against a dark background. Used for living specimens Phase contrast microscopy = light waves that are in phase reinforce one another and their total intensity increases. Light waves that are out of phase by exactly one-half wavelength cancel each other and result in darkness. Contrast is provided by differences in light intensity and as a result the specimen appears as various levels of “dark” against a bright background. Used for living specimens Fluorescence microscopy = uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation Use coarse-focus knob to bring image into focus and use the fine-focus knob to bring the image into sharpest focus. From high dry to oil immersion, should only use fine-focus knobs Microscopic Examination of Eukaryotic Microbes Wet Mount Procedure: 1. If observing a microbe obtained from solid medium, place loopful or a drop of water on slide. If specimen is already in liquid medium, this step is unnecessary. 2. Use an inoculating needle or loop to transfer specimen from solid medium into water and gently mix 3. Gently lower cover slip onto the drop 4. Don’t use oil immersion if there is a cover slip because the lens may hit it 5. If staining, add a drop of stain next to cover slip. Draw the stain under the cover slip with a piece of paper in contact with the cover slip on edge on the opposite side 4 supergroups of Eukaryotes: 1. Archaeplastida (Ex: Volvox - a green alga) 2. SAR (Ex: Diatoms ) 3. Excavata 4. Unikonta (Ex: Amoeba) Microscopic Examination of Freshwater Microbes Cyanobacteria = easily seen without staining because of their photosynthetic pigments which give them a bluish-green color. When they are found in chains, they are called trichomes. Invertebrate animals = examples include nematode worm and planarian (a flatworm). Simple Stains Stain = solution consisting of a solvent (usually water or ethanol) and a colored molecule (=chromogen). The auxochrome is the charged portion of the colored molecule that allows it to act as a dye through ionic or covalent bonds. Because cytoplasm is transparent, cells usually stained with dye to make them more visible. Basic stain = positively charged chromogen attracted to the negative charges on the surface of most bacterial cells (Ex: methylene blue, crystal violet, and safranin). Basic stains are applied to heat-fixed bacterial smears. Negative stain = only the background is stained, helpful for size and shape determination. Making a bacterial smear (=emulsion): 1. Add bacteria (and a drop of water if needed) to slide and allow smear to air dry. If prepared correctly, the smear should be slightly cloudy. 2. Using a slide holder, pass smear through upper part of flame 2-3 times to heat-fix (heat-fixing is the most common preparation). 3. Allow slide to cool and continue with staining protocol → 4. Place slide on a rack over staining tray and cover smear with stain (staining times differ for each stain; overstaining can cause cell wall disruption). 5. Grasp slide with slide holder and hold at angle. Rinse the slide with distilled water. 6. Gently blot dry in bibulous paper and observe in oil immersion. Gram Stain → a differential stain (most common differential stain) → allows detection of differences between organisms or different parts of same organism, used more frequently than simple stain. The Gram stain also allows determination of cell morphology, size, and arrangement, it is typically the first differential test run on a specimen brought into lab for identification. - The primary stain is crystal violet - Iodine is added as a mordant to enhance crystal violet staining by forming a crystal violet-iodine complex - Mordant = combines with a dye or stain to fix it in a material - Decolorization (generally an alcohol/acetone mixture) follows and is the most critical step in the procedure - Gram negative cells are decolorized by the solution whereas Gram positive cells are not - Gram negative cells are therefore colorized by the red counterstain safranin, but Gram positive cells are already violet and cannot - Iodine does not form a complex with safranin - - Eukaryotic cells always stain pink! No cell wall = nothing to trap crystal violet Gram negative cell walls have a higher lipid content (because of the outer membrane) and a thinner peptidoglycan layer than Gram positive. The alcohol/acetone in decolorizer extracts the lipid, making the Gram negative wall more porous and incapable of retaining the crystal violet-iodine complex, thereby decolorizing it The thicker peptidoglycan and greater degree of cross-linking (because of teichoic acids) of Gram positive cells traps the crystal violet-iodine complex more effectively, making the wall less susceptible to decolorization Source of mistake/poor Gram staining: - Overdecolorizing = leaving the decolorizer on too long and get ~reddish Gram positive cells - Undercolorizing = not long enough and get ~purple Gram negative cells ⇒ FALSE results - Inconsistency in preparing emulsion. A good emulsion is about a dime size and dries to a faint haze on the slide - Organisms themselves -- some Gram positives such as Bacillus and Staphylococcus  lose their ability to retain the crystal violet-iodine complex in 24 hours of incubation and can falsely appear all pink Controls: - Staining known Gram positive and Gram negative organisms on either side of you unknown organism act as positive controls - Direct smear made from the gumline can also be used (it will show numerous Gram positive & Gram negative) Acid-Fast Stains (a differential stain) - The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain. Mycolic acid gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. - Primary stain = carbolfuchsin → it is lipid soluble and penetrates the waxy cell wall of acid-fast cells. - Two types of acid-fast procedures: Ziehl-Neelsen (ZN) & Kinyoun (K) method ZN uses heat (steaming) as part of the staining in addition to heat-fix while K is a “cold” stain that still uses heat-fix - Heating melts the mycolic acid and allows the stain to penetrate the cell walls. - Acid alcohol used to decolorize non-acid-fast cells whereas acid-fast cells resist decolorization. - Acid-fast stain is an important differential stain used to identify bacteria in the genus Mycobacterium (ex: Mycobacterium tuberculosis). - Acid fastness is a characteristic that is shared by just a few organisms. Many bacterial cells are easily stained with simple stains or using the Gram stain. Acid fast is useful when acid fast positive bacteria are suspected. Capsule Stain (a differential stain) - Capsules = outside the cell wall of some bacteria; composed of mucoid polysaccharides or polypeptides that repel most stains because of their neutral charge - Capsules increase the virulence in some microbes (ex: Bacillus anthracis →  Anthrax) by making them less vulnerable to phagocytosis - Capsule stain stains around the cells - Typically an acidic stain like Congo red which stains the background and a basic stain that colorizes the cell are used in combination - Capsule remains unstained an appears as a white halo between the cells and the colored background - Capsule staining begins as a negative stain; cells are spread into a film with an acidic stain and not heat-fixed. Heat-fixing causes cells to shrink, leaving an artificial white halo around them that may be interpreted as a capsule - The sample is emulsified in serum instead of heat fixing to hold it on the slide Endospore Stain (a differential stain) - Some bacteria are able to differentiate into dormant cells called endospores when environmental conditions, such as nutrient depletion or high temperatures, are unsuitable for growth - Endospores are highly resistant to heat and chemicals, total absence of ATP in endospores - A tough outer covering made of the protein keratin resists staining so extreme measures must be taken to stain an endospore - In the Schaeffer-Fulton method, primary stain malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material so “vegetative cells” and “spore mother cells” which are responsible for producing the endospore can be decolorized with water and counterstained with safranin - Endospores may be located in the middle of the cell (central), at the end of the cell (terminal) or between the end and middle (subterminal). Endospore location in some species is variable and may be a combination of terminal and subterminal. Endospore characteristics are unique to each species - Endospores may be spherical or elliptical (oval) - Most common species to produce endospores are in the genera Bacillus and Clostridium. All species of Bacillus make endospores - Endospores do not stain but they are visible in a Gram stain - In this lab we used different strains of Bacillus both 48-hour and 5-day (young cultures of spore-forming microbes may not demonstrate any endospores because the vegetative cells may not have been subjected to sufficient stress to stimulate sporulation - If something appears refractive in the cell, it’s not necessarily an endospore - it can be an inclusion (serve as storage vessels in cytoplasm) Wet Mount and Hanging Drop Preparations - Made by placing specimen in a drop of water and covering it with a cover glass (don’t smulsify the specimen with water because risk of damaging flagella) - Since no stain used and cells are transparent, viewing is best with as little illumination as possible by adjusting iris diaphragm - Hanging drop preparation allows longer observation than wet mount because it does not dry out as quickly - Thing ring of petroleum jelly applied around the well of the depression slide causing the cover glass to stick to the slide - Bacteria will swim or they are nonmotile and vibrate in place - The longer you observe a hanging drop, the more energy is produced by the water allowing microorganisms to move around Flagella Stain (read only) - Typically flagella too thin to be observed with light microscope and ordinary stains - Flagella stains use a mordant to assist in encrusting flagella with stain to visible thickness - If bacteria are motile, they have flagella Streak Plate Methods of Isolation - Obtaining isolation of individual species from a mixed sample is generally the first step in identifying an organism. Streak plate is a commonly used isolation technique - A bacterial sample is streaked over the surface of plated agar medium. During streaking, cell density decreases, eventually leading to individual cells being deposited separately on agar surface. Cell that have been sufficiently isolated will grow into separated colonies consisting only of the original cell type - A portion of isolated colony can be transferred to a sterile medium to start a pure culture 1. Inoculation of agar plates using the quadrant streak method (4 streakings) Suspected high cell density 2. Inoculation of agar plates using the T-streak method (3 streakings) 3. Zigzag (continuous) inoculation of agar plates using a cotton swab Typically when not high cell density and pure cultures when isolation not necessary 4. Inoculation of agar plates with a cotton swab in preparation for a quadrant streak plate Suspected high density and isolation of two or more bacterial species in a mixed culture First use the swab to perform first streak, then continue the quadrant streak using loop Spread Plate Method of Isolation - Another method to isolate a pure culture - Small volume of diluted microbial sample is deposited on agar and spread uniformly across the surface with a glass rod → with properly diluted sample, cells will be deposited far enough apart to grow into individual colonies - After incubation, a portion of isolated colony can be transferred to sterile medium to begin a pure culture Ubiquity of Microbes (ubiquity = being everywhere) - In this lab we sampled and cultured several locations in the lab including the desk, hands, and fingers - We also incubated uninoculated plates to serve as a control. If growth appeared on the uninoculated plates, that meant they were contaminated between the autoclave and time of incubation Colony Morphology - Color, size, shape, and texture of microbial growth are determined by the genetic makeup of the organism, but are also influenced by environmental factors like nutrient availability and temperature - Colony characteristics include: Shape - round, irregular, punctiform (tiny, pinpoint) Margin - smooth, undulate/irregular (wavy), lobate, filamentous, or rhizoid Surface - smooth, rough, wrinkled Texture - moist, mucoid (sticky; make CAPSULES), butyrous (buttery), dry, shiny, dull Elevation - convex, umbonate (raised in center), flat, raised - Colony recognition is often the first indication that one organism is different from another - For most colony morphology we identify, the medium used is a general all-purpose nutrient agar which can grow many types of organisms. - *exam question* - why is this bacteria yellow? Answer: because it makes that pigment Growth Patterns on Slants - Most organisms on slants produce filiform growth (dense and opaque with a smooth edge) - Slants are not definitive by themselves, but can provide useful information when identifying organism Growth Patterns in Broth - Some organisms float on top of the medium and produce a surface membrane - Other sink to the bottom as sediment - Some produce uniform fine turbidity