Lab Practical I Study Guide PDF

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You are responsible for all information from Week 1 through Week 3 and any information contained in quiz questions and Mastering Microbiology. You must write in complete sentences with correct spelling. Week 1: Attendance and Safety Policies

2-1 Ubiquity of Microorganisms Applying the terms ubiquity, pathogenic, reservoir, opportunistic pathogen, and saprophyte. Understand the ubiquitous nature of microorganisms. - Ubiquity: microorganisms are ubiquitous in nature, meaning they are very common and are found in a wide range of habitats - Pathogenic: microorganisms that cause damage to their host cell (i.e. cause disease) - Reservoir: any area, including sites outside of the host organism, where a microbe resides and serves as a potential source of infection - Opportunistic Pathogen: microorganism capable of producing a disease state if introduced into a suitable part of the body - Saprophyte: microorganism that decomposes organic matter Know what a microorganism must acquire from its environment in order to grow and replicate. - Microorganisms must acquire adequate nutrients and a suitable temperature to grow and replicate Understanding why morphological characterization is useful. - An ability to recognize differences in colony morphology is often the first clue that two organisms are different species. - First indication that two organisms are different Understanding why microbial controls (germicides) are useful. - Prevent infections - They are useful b/c germicides kill microorganisms and inactivates viruses

Exercise 2-2: Colony Morphology -

Colony = a visible mass of cells Color, size, shape, and texture of microbial growth are determined by the genetic makeup of the organism, but are also greatly influenced by environmental factors including nutrient availability, temperature, and incubation time. Applying The 5 Morphology Categories and the descriptive words used for each category (in bold in your lab manual).

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Shape: round, irregular, or punctiform (tiny, pinpoint) Margin: o Entire (smooth, w/ no irregularities) o Undulate (wavy) o Lobate (lobed) o Filamentous (unbranched strands) o Rhizoid (branched like roots) Surface: smooth, rough, wrinkled Texture: o Moist o Mucoid (sticky) o Butyrous (buttery) o Dry o Shiny o Dull

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Elevations: o Flat o Raised o Convex o Pulvinate (very convex) o Umbonate (raised in the center) Color & Optical Properties: opaque (can’t see thru it) and translucent (light passes thru)

Distinguishing morphology types if provided images found in Fig. 2-3.

Exercise 2-3: Growth Patterns on Slants -

Agar slants are useful primarily as media for cultivation and maintenance of stock cultures Two important factors influencing microbial growth are incubation time and temperature

Applying the terms friable, filiform, and spreading edge. - Filiform – smooth texture w/ solid edge - Spreading edge – solid growth seeming to radiate outward - Transparent – almost invisible, or easy to see light thru - Friable – rough texture w/ crusty appearance - Pigmented – produces colored growth

Exercise 2-4: Growth Patterns in Broths -

Bacterial genera, and frequently individual species w/in a genus, demonstrate characteristic growth patterns in broth that provide useful information when attempting to identify an organism Applying the terms pellicle, sediment, uniform fine turbidity, and flocculence. - Flocculent – suspended chunks or pieces - Sediment – growth on the bottom - Ring – growth at top around the edge - Pellicle – membrane at the top - Uniform fine turbidity – evenly cloudy throughout

Pg 121: Applying the term germicide. - Germicide = an agent that kills microorganisms and inactivates viruses Applying the three broad germicidal categories: decontamination, disinfection, and sterilization. - Decontamination – lowest level of control – the reduction of pathogenic microorganisms to a level at which items are safe to handle w/o protective attire – includes physical cleaning w/ soaps/detergents, and removal of all (ideally) or most organic material - Disinfection – the next level of control (divided into 3 sublevels: low, medium, and high – based on effectiveness against specific control pathogens and their surrogates) – all sublevels kill large numbers, if not all, of the targeted pathogens but typically do not kill large numbers of bacterial endospores – disinfectants are typically liquid chemical agents - Sterilization – the complete elimination of viable organisms including bacterial endospores – the highest level of pathogen control – can be achieved by some chemicals, some gases, incineration, dry heat, moist heat, etc.

Exercise 2-12: Steam Sterilization Purpose of an autoclave and description of how it works. - Autoclave = Steam sterilizer - Used for sterilizing surgical/dental instruments, microbiological media, etc. - Autoclaves use super-heated steam under pressure to kill heat-resistant organisms

- 121 C -- items being process must reach optimum temperature for at least 15 minutes How to test autoclave functionality and how to interpret results from the test. - (1) Autoclave Tape – commonly used method of verifying that sterile conditions have been achieved in an autoclave is to place a strip of autoclave tape on the item(s) to be sterilized. Prior to autoclaving, autoclave tape looks very much like masking tape. However, if 121 Celsius for 15 minutes has been produced inside the autoclave, black stripes appear on the tape. - (2) Autoclave Biological Indicators – vial containing spores capable of withstanding high heat. If color changes, not killed. If purple, no viable organisms.

Exercise 5-25: Blood Agar

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Several species of Gram (+) cocci produce endotoxins called hemolysins, which are able to destroy RBCs and hemoglobin - Hemolysins produced by streptococci are called streptolysins o 2 forms:  Streptolysin O – oxygen labile and expresses maximal activity under anaerobic conditions  Streptolysin S – oxygen stable but expresses itself optimally under anaerobic conditions as well Why is blood agar used? - Blood agar is used for isolation and cultivation of many types of fastidious bacteria - It is also used to differentiate bacteria based on their hemolytic characteristics What does the bacteria look like on the media? What do α, β, and γ hemolysis look like? - Alpha hemolysis – partial destruction of RBCs – produces a greenish discoloration of the agar around the colonies - Beta hemolysis – complete destruction of RBCs and hemoglobin – results in a clearing of the medium around the colonies - Gamma hemolysis – is actually non-hemolysis – appears as simple growth w/ no change to the medium Week 2: Understanding why techniques to isolate single colonies are useful. - To be able to observe species Understanding why aseptic techniques are useful. - To be able to transfer microbes w/o contamination

Understanding why calibrating an ocular micrometer is useful. - To be able to measure organism

Exercise 1-3: Common Aseptic Transfers and Inoculation Methods -

Culture = a medium that contains living microbes Pure culture = a culture containing a single species It is essential to transfer microbes from their pure culture to a sterile medium aseptically, that is, w/o contamination of yourself, others, the environment, the source culture, of the medium being inoculated. - Broths are used to grow microbes when fresh cultures or large numbers of cells are required - Agar slants are generally used to grow stock cultures that can be refrigerated after incubation and maintained for several weeks - Plated media are typically used for obtaining isolation of a species, differential testing, and quantifying bacterial densities - The primary concern w/ BSL-2 organisms is contamination or infection due to inhalation of aerosols – aerosols are problematic b/c we generally are unaware of their production and they remain suspended in the air long after the procedure has been completed Proper loop sterilization technique. - 1.16 Flaming Loop – incineration of an inoculating loop’s wire is done by passing it through the tip of the flame’s inner cone. Begin at the wire’s base and continue to the end, making sure that all parts are heated to a uniform orange color. Allow the wire to cool before touching it or placing it on/in a culture. The former will burn you; the latter will cause aerosols of microorganisms. Proper transfer technique for: slant to slant/broth broth to slant/broth plate to slant/broth -

SLANT TO SLANT/BROTH 1. Flame inoculating loop 2. Remove cap off inoculant tube and flame mouth of tube 3. Hold tube at an angle and use inoculating loop to pick up culture 4. Flame tube and replace lid 5. Remove cap of sterile culture and flame tube 6. Inoculate by starting at the base and gently move/inoculate by gently swirling the loop in the broth 7. Flame tube and recap 8. Sterilize loop in flame

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BROTH TO SLANT/BROTH 1. Flame loop 2. Remove cap off inoculant tube and flame tube 3. Hold tube at an angle and use loop to pick up culture 4. Flame tube and replace lid 5. Remove cap of sterile culture and flame tube 6. Inoculate by moving the tube to the tip of the sterile medium starting at the base and gently move the loop back and forth as you remove the slant/ inoculate by gently swirling the loop in the broth and remove the broth from the loop 7. Flame tube and recap 8. Sterilize loop in flame

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PLATE TO SLANT/BROTH 1. Flame loop 2. Lift lid of plate, but continue to use it to cover to prevent contamination

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Touch loop to inoculate portion to cool it Obtain a small amount of growth by gently touching a colony w/ the wire tip Remove loop from plate and hold it still as you replace the lid Inoculate by starting at the base and gently move/inoculate by gently swirling the loop in the broth – the loop bac and forth up the slant 7. Flame tube and recap 8. Sterilize loop

Understanding the information associated with Figures 1-9 through 1-23. Physically repeating this procedure.

Exercise 1-5: Spread Plate Method of Isolation Proper spread plate technique. - The spread plate technique is a method of isolation in which a diluted microbial sample is deposited on an agar plate and spread uniformly across the surface w/ a glass rod – w/ properly diluted sample, cells will be deposited far enough apart on the agar surface to grow individual colonies Understanding the information associated with Figures 1-39 through 1-42. Physically repeating this procedure.

Supplement Page 5: Pour Plate Technique Proper pour plate technique. - The pour plate technique is one of many techniques used to obtain isolated colonies of bacteria and fungi - The original sample is diluted several times to sufficiently reduce the microbial population in order to yield separate colonies upon plating. - Small volumes of ea. dilution are mixed w/ molten tryptic soy agar and then poured into sterile petri plates - After the agar has solidified, ea. isolated cell will proliferate and form a colony – Isolated colonies will result when the sample is diluted to a point that the cells are separated enough in the agar to make individual colonies - The total number of colonies is equivalent to the number of viable microorganism in the diluted sample -

Used to obtain isolated colonies of bacteria and fungi – the original sample is diluted several times to sufficiently reduce the microbial population

Understanding how to create a dilution scheme. - Serial dilution – a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration – ea. dilution will reduce the concentration of bacteria by a specific amount Understanding the calculations behind a dilution scheme: (e.g. 0.1mL of culture into 9.9mL of saline creates a 1 in 100 dilution (1mL of culture for every 100 mL of volume), or 10-2. Furthermore, 1mL of the 10-2 dilution put into 9mL of saline creates a 1 in 1000 dilution (1mL of culture for every 1000mL of volume), or 10-3, etc.). Physically repeating this procedure.

Exercise 1-4: Streak Plate Methods of Isolation -

Mixed culture – a microbial culture consisting of two/more species Pure culture – a microbial culture containing only a single species Obtaining isolation of individual species from a mixed sample is generally the first step in identifying an organism – commonly used isolation technique = streak plate The identification process of an unknown microbe relies on obtaining a pure culture of that organism – the streak plate method produces individual colonies on an agar plate – a portion of an isolated colony then may be transferred to a sterile medium to start a pure culture

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In the streak plate method of isolation, a bacterial sample (always assumed to be a mixed culture) is streaked over a surface of a plated agar medium. o During streaking, the cell density decreases, eventually leading to individual cells being deposited separately on the agar surface o Cells that have been sufficiently isolated will grow into colonies consisting only of the original cell type

Proper streak plate technique. - All streak patterns are designed to separate deposited cells on the agar surface so individual cells grow into isolated colonies o a quadrant streak (T-streak) is generally used w/ samples suspected of high cell density o simple zigzag (continuous streak) pattern may be used for samples containing lower cell densities Understanding the information associated with Figures 1-34A through 1-34D. - 1.34ABCD – o Beginning the Quadrant Streak Pattern – Streak the mixed culture back and forth in one quadrant of the agar plate. Stay close to the plate’s edge and make the streaks long. Do not cut the agar w/ the loop. Flame the loop and then proceed o Second Streak – Rotate the plate nearly 90 degrees and touch the agar in an uninoculated region to cool the loop. Streak again, using the same wrist motion. Flame the loop afterward. o Third Streak – Rotate the plate nearly 90 degree and streak again. Be sure to cool the loop prior to streaking and flame it afterward. o Fourth Streak into the Center – After cooling the loop, streak one last time into the center of the plate. Flame the loop and incubate the plate in an inverted position for the assigned time at the appropriate temperature. Physically repeating this procedure. Week 3:

Exercise 3-1 and 3-2: Introduction to Light Microscope and Calibration of Ocular Micrometer -

Ocular micrometer – type of ruler installed in a microscope eyepiece, composed of uniform but unspecified graduations – it must be calibrated before any viewed specimens can be measured - Cell dimension = ocular units X calibration How to calibrate an ocular micrometer. - The device used to calibrate an ocular micrometer is called a stage micrometer How to take measurements with an ocular micrometer.

Calculations (including unit conversions) associated with ocular micrometer calibrations.

1Ocular Space=

¿ spaceson stage micrometer × 10 μm ¿ spaces on ocular micrometer

Understanding why staining techniques are useful. Suggest a staining reagent(s) used in lab that could be used with each type of stain.

Exercise 3-8 Acid-Fast Stains -

Purpose of Acid-Fast Stains: the acid-fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated w/ an acid alcohol – it is an important differential stain used to identify bacteria in the genus Mycobacterium

Understanding what component of cell walls causes a microorganism to be considered acid-fast. - The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain Understanding how and why the stains interact with a cell. - Mycolic acid – a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution - The waxy wall of acid-fast cells repels typical aqueous stains (as a result, most acid-fast positive organisms are only weakly Gram positive) Distinguishing between a positive and negative result. - Acid-fast cells stain reddish-purple - Acid-fast negative cells stain blue (from methylene blue) or the color of the counterstain if a different one is used Theoretically performing this procedure. - Ziehl-Neelsen Acid-Fast Stain Overview: 1. Cells prior to staining are transparent 2. After staining w/ carbolfuchsin, cells are reddish-purple. Steam heat enhances the entry of carbolfuchsin into cells 3. Decolorization w/ acid alcohol removes stain from acid-fast negative cells 4. Methylene blue is used to counterstain acid-fast negative cells - Kinyoun Acid-Fast Stain Overview: 1. Cells prior to staining are transparent 2. After staining w/ carbolfuchsin, cells become reddish-purple. NO steaming is necessary 3. Decolorization w/ acid alcohol removes stain from acid-fast negative cells 4. Methylene blue is used to counterstain acid-fast negative cells

Exercise 3-10 Endospore Stain - Purpose of Endospore Stain: the endospore stain is a differential stain used to detect the presence, shape, and -

location of endospores in bacterial cells Endospores may be differentiated based on shape – either spherical or elliptical (oval) – and size relative to the cell, that is, whether they cause the cell to look swollen or not

Applying the terms endospore, vegetative cell, and spore mother cell. - Some bacterial species are able to differentiate into dormant cells called endospores when environmental conditions, such as nutrient depletion or high temperatures, are unsuitable for growth o Endospores are highly resistant to heat and chemicals, which allows them to survive in this dormant state for long periods of time o Endospores resistance is due to a combination of factors, including a tough outer covering made of the protein keratin, its dehydrated state, DNA protective proteins, and other adaptations o In addition to nutrient depletion, sporulation has also been shown to be dependent on population density

- Endospore – dormant cell that appears when environmental conditions are unsuitable for growth - Vegetative cells = metabolically active cells - Spore Mother Cell = responsible for producing the endospore - When conditions are suitable again, endospores germinate into metabolically active vegetative cells Applying the spore location terms central, terminal, and subterminal. - Endospores located in the middle of cell = central - Endospores located at the end of cell = terminal - Endospores located between the end and middle of the cell = subterminal Understanding how and why the stains interact with a cell. - The keratin in the spore coat also resists staining, so extreme measures must be taken to stain an endospore - In the Schaeffer-Fulton Method, a primary stain of malachite green is forced into the spore by steaming the bacterial emulsion – alternatively, malachite green can be left on the slide for 15 minutes or more to stain the spores – malachite green is water soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized w/ water and counterstained w/ safranin Distinguishing between a spore and a vegetative or spore mother cell. - Spore Mother Cell – cells responsible for producing the endospore - Spore – a specialized/differentiated cell that is highly resistant to desiccation and heat Physically repeating this procedure. - 3.111 The Schaeffer-Fulton Endospore Stain: Upon completion, endospores are green, vegetative and spore mother cells are red 1. Cells and spores prior to staining are transparent 2. After staining w/ malachite green, cells and spores are green. Heat is used to force the stain into spores in present. 3. Decolorization w/ water removes stain from cells, but not spores 4. Safranin is used ...