Microbiology Lab 3051 (Spring 2016)
Mid-Term Laboratory Exam
Labs 1 - 5
1. Know the parts of the microscope and their functions.
Arm and base: basic frame structure which all other parts are attached to Stage: horizontal platform that support the microscope slide If you want to learn more check out Why is stoicism wrong?
Staging adjustment: used for holding and moving the slide around on the stage Light adjustment control: vary the intensity of light
Ocular: eyepiece, located at the top of the instrument, consists of two internal lenses that add magnification of 10x (there are two oculars) Don't forget about the age old question of Where does junctional diversity occur?
Objectives: various magnification lens (scanning, low-power, high-dry, oil immersion)
Nosepiece: 3 or more objective lens are attached to this piece
Condenser: located under the stage, collects and directs the light from the lamp to the slide being studied, does not affect the magnifying power, can be moved up and down
Diaphragm: within the condenser regulates the amount of light that reaches the slide
Course adjustment: focusing knobs for lower power magnification
Fine adjustment knobs: high power focusing knobs
2. Know how to use the microscope (adjusting for proper viewing, getting an image in focus, using oil immersion) and how to take care of a microscope. • Place slide on stage
• Reposition so stained material is in the center of the light source • Ensure condenser is raised to its highest point We also discuss several other topics like What does long term potentiation do?
• Bring into focus using coarse adjustment knobs on lower magnification • Turn nosepiece to high magnification
• Use fine coarse adjustment knobs to refocus
• Manipulate diaphragm to reduce or increase the light intensity • If using oil immersion, turn nosepiece, add a drop of oil and then turn to oil immersion lens – make sure you do not use a non-oil immersion lens with oil • Move the slide using the stage adjustment knobs to search for what you are looking for
• Be sure the clean of lens using a cleaning tissue if you used oil • When putting away return nosepiece to scanning lens
• Replace dustcover
Parfocal: image remains in focus when changing from a lower-power objective lens to a high-power lens
3. Describe aseptic technique and understand why it is important. Aseptic Technique
Work Area Disinfection
• Work area is treated with disinfectant to kill any microorganisms that may be present If you want to learn more check out What are the symptoms of depression?
We also discuss several other topics like What are the related principles of classical and operant conditioning?
Loops and Needles
• Loops and needles are sterilized in a Bunsen burner flame until it is red hot
• Incinerates any contaminating organisms
• Allow to cool before picking up bacteria
Culture Tube Flaming and Inoculation
• Cap is removed and held with the pinky
• Mouth of the tube is flamed
• Tube is inoculated with bacteria (broth-loops swirled in broth several times; slant-loop pulled from bottom to top of agar; stab-needle is stabbed into agar; petri dish-plate is raised and held diagonally to protect the surface from any contamination in the air)
• Mouth of tube is reflamed after inoculation and cap is replaced Final Flaming of Loop or Needle
• After inoculation, tool is resterilized and returned to storage – not work desk
Final Disinfection of the Work Area
• When all work is complete, the work area is retreated with disinfectant
Steps for inoculating broth culture with inoculating loop:
1. Inoculating loop is heated until it is red-hot We also discuss several other topics like What is performance management?
2. Organism in culture are dispersed by shaking tube
3. Tube cap is removed and mouth of tube is flamed
4. A loopful of organism is removed from tube
5. Loop is removed from culture and tube mouth is flamed
6. Tube is returned to tube
7. Cap for nutrient broth is removed and tube is flamed
8. Loop with bacteria is inserted into tube of sterile broth
9. Loop is removed from broth and tube mouth is flamed
10. Broth tube is recapped
11. Loop is flamed and returned to receptacle
Importance: use of aseptic technique insures that no contaminating organism are introduced into culture material when the latter are inoculated or handled in some manner. Also insures that organisms that are being handled do not contaminate the handler or others who might be present.
4. Compare and contrast how to make a bacterial smear from liquid media and solid media.
Make a bacteria smear from liquid media:
• Draw “target circle” on bottom of slide
• Two loopfuls of liquid containing organisms are placed in the center of the “target circle”
• Organisms are dispersed over entire area of the “target circle” • Smear is allowed to dry
• Slide is passed through flame to heat kill and fix organisms
Make a bacteria smear from solid media:
• Draw a “target circle” on bottom of slide
• Two loopfuls of water are placed in center of “target circle”
• A very small amount of organisms is dispersed with inoculating loop in water over entire are of “target circle”
• Smear is allowed to dry
• Slide is passed through flame to heat kill and fix organism
Compare: both should result in similar number of bacteria on the slide, both are dried and heat fixed
Contrast: bacteria growing on solid media tend to cling to each other and therefore, must be dispersed sufficiently by dilution in water
5. Explain the importance of heat fixing and air-drying specimens on slides. Heat fixing and air-drying ensures that cells adhere to the microscope slide and are not washed off during subsequent staining and washing procedures
6. Distinguish between acidic and basic dyes and when to use each. Basic dyes have color bearing ions (chromophores) that are positively charged (cationic). Basic dyes are used to stain most bacteria because most bacteria are negatively charged and therefore attracted to the basic dye.
Acidic dyes have chromophores that are anionic and they bind to bacteria that haven positive charged surfaces. Most bacteria will repel acidic stain.
7. Describe simple staining and what bacterial characteristics can be observed using this technique.
Simple staining is the use of a single stain to color a bacterial cell. Simple staining can be used to assess the size, shape, form, and arrangement of cells.
8. Recognize the different bacterial morphologies.
• Cocoobacillus: short rods
• Bacilli: several rods in a row
• Fusiform: rods spread out
• Diplococci: cocci doubles
• Tetrads: cocci groups of four
• Streptococci: cocci chains
• Staphylococci: cocci in grape-like clumps
Spiral or Curved
• Comma: comma shaped bacteria with spiral sticking out
• Spirilium: wavy bacteria with spirals sticking out
• Spirochaetes: long thin spirals
9. Understand the distribution of bacteria in our world.
10. 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: thick outer layer of peptidoglycan right outside of the cell membrane; stains dark purple
Gram negative cells: thin layer of peptidoglycan between periplasmic space outside the cell membrane and an outer membrane; stains pink
11. 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.
• Heat-fix bacteria to the plate
• Cover smear with crystal violet (primary stain)
o Crystal violet binds to peptidoglycan layer in gram positive, and outer membrane in gram negative
o Both bacteria are stained purple at this point
• Briefly wash off the stain with distilled water
• Cover the smear with Gram’s iodine (mordant) solution
o Iodine acts as a mordant, that forms an insoluble complex with crystal violet in gram positive cells
o Both bacteria are still purple
• Wash off iodine with 95% alcohol (decolorizing agent) until the alcohol flows away colorless
o Outer membrane in gram negative cell is removed from gram negative cells, alcohol does not decolorize gram positive cells
o Gram positive remain purple, gram negative are now colorless • Wash with wash
• Cover with safranin (counterstain)
o Safranin binds to peptidoglycan layer that is now exposed in gram negative cells, and peptidoglycan layer in gram positive
o Gram negative are now pink, gram positive are a slightly darker purple
• Wash for a few seconds, blot dry, examine under oil immersion
12. Understand the purpose of a streak plate and how you would do one. The purpose of a streak place is to obtain a pure culture. Pure cultures contain only a single kind of an organism. With a pure culture we are able to study the cultural, morphological, and physiological characteristic of an individual organism.
Quadrant Streak procedure: divide the petri dish into 4 quadrants • Streak one loopful of organisms back and forth over Area 1
• Flame the loop, then cool for 5 seconds
• Rotate dish 90 degrees while keeping dish closed, streak area 2 with several back and forth strokes, hitting the original streak a few times
• Flame the loop again, repeat last step in quadrant 3
• Repeat again in quadrant 4 – at the end drag the loop into the middle of the culture
• Flame loop and put it aside
The point of streaking is to end up with a single colony
13. Define pure culture and pure colony.
A pure culture contains only a single kind of organism. A pure colony is a colony that has arisen from a single cell.
14. Know the purpose of a gelatin stab, how you conducted this test, and what a positive reaction for protease production would look like.
Some bacteria produce proteases, proteins that degrade proteins. To determine if an unknown produces proteases, inoculate an agar by inserting a needle 2/3 of the way into the tube. Leave tube in refrigerator or freezer. If material is liquefied, the bacteria contains proteases (positive), if it is solid, the bacteria does not produce proteases (negative). Some bacteria may also be able to grow in the gelatin, however this property and formation of the growth is not particularly important or relevant to the test results.
15. Describe endospores, their function, their unique characteristics, and which genera produce them.
When species of bacteria belonging to the genera Bacillus or Clostridia run out of essential nutirents, they undergo a complex developmental cycles that produces a resting stages called endospores. Endospores are very dehydrated and are not actively metabolizing. They are resistant to heat, radiation, acids, and many chemical that normally harm or kill vegetative cells. They have a protein coat around them that serves as a protective barrier. Endospores can exist in their state for a long time until nutrients become available again and they undergo a process of germination to form a new vegetative cell that will resume normal growth.
16. Understand the Schaeffer-Fulton endospore staining procedure (know the primary stain and counterstain), why heat is used as a mordant, and what endospore producers look like under the microscope after being stained.
Endospore are not easily penetrated by stains. In SF method heat is applied while staining with malachite green (primary stain). The malachite green penetrates the endospore during heating, but is not removed during decolorization. The heat acts as a mordant to facilitate the uptake of the stain. Safranin is used as the
counterstain and stains the vegetative portion of the cell. When examined under the microscope, you will see a green endospore contained in a pink sporangium.
17. Know what genera of bacteria are acid-fast and what structure causes them to be acid-fast.
Bacteria such as Mycobacterium and some Nocardia have cells walls that contain high lipid content. One of these lipids is a waxy material called mycolic acid. This lipids prevents bacteria from being stained by many of the stain routinely used in microbiology.
18. Describe the acid-fast stain (primary stain, decolorization, counterstain) and know how acid-fast cells and non-acid fast cells appear after staining.
In acid-fast staining heat is used as mordant to make the stain complex more permeable to the mycolic acid and cell wall lipids. For Ziehl-Neelsen method the primary stain is carbolfuchsin. Cells are heated and carbolfuchsin penetrates into the cell. Acid-alcohol is used as a decolorizer and removes primary stain from non-acid fast cell. Stain is trapped inside the lipid cell wall is acid-fast cell. The counterstain, methylene blue is then used to stain the non-acid fast bacteria. Under microscope, acid-fast bacteria retain primary stain and appear pink/red, non-acid-fast bacteria appear blue.
19. Describe bacterial capsules and their functions.
Bacteria capsules are a layer of polysaccharides that resides outside the cell envelope. Capsules help protect the cell and make it more difficult for our immune system to destroy them.
20. Describe the use of negative staining to visualize capsules and know how capsules appear after using this method.
• Negative stains are acidic and therefore negatively charged. The cell repels negative stains.
• This results in the background surround the cell being colored while the cell in the center is colorless. Cells appear as transparent objects against a dark background.
• Negative stains can be useful in characterizing some of the external structures, such as capsules. Capsules appear as a halo surrounding a positively stained cell against a dark background.
• No heat fixation is performed so cell size and shape can be determined more accurately.
21. Understand the methods we used to see motility and how motility (or lack of) was determined.
• Major organelle used for motility in bacteria are flagella, Flagella move using a process called chemotaxis. Flagella are very thin structures, must be stained using special techniques in order to be observed.
• Motility and the arrangement of flagella around the cell are important taxonomic characteristics that are useful in characterizing bacteria • Wet mount: a drop of viable cells is placed on a microscope slide and covered with a cover glass. The slide is then observed with a phase contrast microscope. The rapid swimming movement of cells in the microscopic field confirms motility.
• Hanging drop technique: a drop of cells is placed on a cover glass, which is then placed over a special slide that has a concave depression in its center. The coverslip is held in place with petroleum jelly. Observe under microscope using high-dry power and focusing on the edge of the drop. Cells observed to be moving are motile.
22. Of the bacteria we used for the motility tests, be able to name which bacteria are motile and non-motile.
Proteus vulgair = motile
Micrococcus luteus = non motile
23. 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).
Purpose: determine if bacteria is capable of fermenting sugars suchas glucose, lactose, or mannitol
Positive results: yellow color, may have gas in Durham tube
Media: glucose with Durham tube
Summary: if an organism ferments a sugar, acid is usually produced, sometimes along with gas; presence of acid causes a color change in the pH indicator, phenol red, from red at alkaline pH values to yellow at acidic pH values; presence of gas is revealed by the displacement of medium from the Durham tube
Mixed Acid Fermentation (MR)
Purpose: Determine if bacteria perform mixed acid fermentation. Some gram negative bacteria ferment glucose to produce a number of organic acids such as lactic, acetic, succinic, and formic acid. In addition CO2, H2, and ethanol are also produced in this fermentation. A sufficient amount of acid is produced it will lower the pH of methyl red to 5 or less.
Positive results: red color
Reagents: methyl red
How reagents work: pH indicator, methyl red, is added to the medium, which turns red if acid is present. A positive methyl red test indicates that the organism has carried out mixed-acid fermentation. Bacteria that are mixed-acid fermentators also produce gas.
Butanediol Fermentation (VP)
Purpose: Determine if gram-negative bacteria carry out butanediol fermentation. Some bacteria ferment glucose to produce limited amounts of some organic acids and primarily a more neutral end product, 2, 3 butanediol.
Positive results: red color
Reagents: Barritt’s A reagent, Barritt’s B reagent
Summary: 2,3 butanediol is not detected directly but must be converted to acetoin by oxidation of 2, 3 butanediol. Acetoin reacts with Baritts agent. Reagent is
added to a 3 to 5 days old culture. MR-VP medium is shaken; tube will turn pink to red if acetoin is present.
Purpose: Determine if bacteria is capable of using citrate as a sole carbon source. Normally citrate is oxidatively metabolized by Kreb’s cycle, but some bacteria can cleave citrate to produce oxaloacetate and pyruvate. These are fermented to produce formate, acetate, lactate, acetoin, and CO2
Positive results: blue color
Media: citrate agar slate
Summary: Organisms degrade citrate and ammonium salts in the medium and produce ammonia that causes the medium to become alkaline. Under alkaline conditions, the p indicator in the medium turns from dark green to a deep blue, indicating utilization of citrate.
Purpose: Test for presence of cytochrome oxidase, an enzyme in the electron transport chain. This enzyme catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen, producing oxidized cytochrome c and water. Cytochrome oxidase occurs in bacteria that carry out respiration where oxygen is the terminal electron acceptor
Positive results: purple/black color
Media: TSA plate
Reagents: oxidase reagent
Summary: Test differentiates between bacteria that have cytochrome oxidase and use oxygen as terminal electron acceptor from those that can use oxygen as a terminal electron acceptor but have other types of terminal oxidases. Enzyme is detected by the use of an artificial electron acceptor, which changes from yellow to purple when electrons are transferred from reduced cytochrome c to the artificial acceptor.
Purpose: Differentiate strict anaerobes from aerobes or facultative aerobes Positive results: vigorous bubbling
Media: nutrient agar slant
Reagents: hydrogen peroxide
Summary: Aerobic bacteria grow by respiration. During respiration they use oxygen as a terminal electron acceptor. They produce hydrogen peroxide as a by product, however hydrogen peroxide is very damaging to the cell so aerobes produce enzyme catalase, which degrades H2O2. Strict anaerobes lack catalase. To determine if catalase is produced, hydrogen peroxide is added to the specimen. If catalase is produced, there will be vigorous bubbling. If no bubbling occurs, the bacteria lack catalase.
Media: nitrate reduction broth with Durham tube
Reagents: reagants A and B, zinc dust
Summary: Some facultative anaerobes can use nitrate as a terminal electron acceptor in nitrate respiration. Some bacteria reduce nitrate to gaseous products or nitrite. One of the enzymes involved in this process is nitrate reductase. This enzyme is only produced if nitrate is present and anaerobic conditions exist for growth.
Purpose: Determine if bacteria performs starch hydrolysis to break down starch macromolecules
Positive results: Clear zone along streak
Media: Starch agar
Reagents: Gram’s iodine
Summary: Bacteria that hydrolyze starch produce amylases that degrade the starch molecule into smaller molecules. Starch hydrolysis is detected by adding iodine to starch medium. Iodine complexes with the starch macromolecule and causes the medium to turn blue. If starch has been degraded, the medium adjacent to the bacterial growth will be clear after the addition of the iodine
Purpose: Determine if bacteria is capable of hydrolyzing casein (proteins) Positive results: Clear areas adjacent to growth
Media: Skim milk agar
Summary: Many bacteria produce proteases, which are enzymes that degrade protein molecules. If proteases are present in the bacteria, it will be able to perform hydrolysis to break down casein, a protein found in milk, leaving a clear zone on the skim milk agar
Purpose: Determine if bacteria is capable of hydrolyzing tryptophan to produce indole
Positive results: Red ring on the surface of the broth
Media: Tryptone broth
Summary: Some bacteria have the ability to degrade amino acid tryptophan to produce tryoptophan using enzyme trypotphanase. The degradation of tryptophan can be detected using Kovac’s reagent, which forms a deep red color if indole is present
Purpose: Determine if bacteria can hydrolyze urea
Positive results: cerise color
Media: urea area slant
Summary: Urea is a waste product of animal metabolism that is broken down by a number of bacteria. The enzyme responsible is urease, which splits the molecule into carbon dioxide and ammonia. When urease is produced by a bacteria, the resulting ammonia causes the pH to become alkaline. As pH increases, phenol red changes from yellow to a bright pink or cerise color
24. 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.
• Functional type: selective and differential
• Mechanism: Bile salts and crystal violet prevent gram-positives form growing • Type of colonies: gram-negatives
• Distinguishing: Lactose fermenter turn pH indicator pink because they produce acid; non-lactose fermenters don’t turn the plate pink
• Functional type: selective
• Mechanism: phenylethyl alcohol prevent gram-negatives from growing • Type of colonies: gram-positives
• Distinguishing: none
• Functional type: enriched and differential
• Mechanism: none
• Type of colonies: both gram-negative and gram-positive
• Distinguishing: allows for differentiating based on ability to produce hemolysins - proteins that lyse red blood cells; A-hemolytic cell partially degrade blood cells and turn greenish color, B-hemolytic cells completed break down red blood cells and turn clear color; Gamma-hemolytic cells do not have hemolysins and remain regular colors
25. Understand how to construct a bacterial growth curve by measuring the turbidity of a broth culture.
26. Know the four phases of bacterial growth in a batch culture.
• Death or decline
27. Be able to classify organisms based on their oxygen requirements (obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, aerotolerant) as shown by growth in fluid thioglycollate medium (FTM).
• Obligate aerobes – must be grown in oxygen, oxygen is terminal electron acceptor
• Obligate anaerobes – cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated, otherwise they are harmed or killed by its presence
• Facultative anaerobes – grow very well aerobically but also have the capacity to grow very well aerobically but also have the capacity to grow anaerobically if oxygen is not present byswitching to fermentation
• Microaerophiles – aerobic bacteria that prefer to grow in low concentration of oxygen (2%-10% as opposed to atmosphere’s 20%)
• Aerotolerant (Obligate fermenters) – can tolerate oxygen and grow in its presence but they do not require oxygen for energy production, rely solely on fermentation
Growth in FTM:
28. Define neutrophile, acidophile, and alkaliphile.
• Neutrophile: bacteria that grow at or near neutral pH
• Acidophile: bacteria that grow at acidic pH values
• Alkaliphile: bacteria that grow at basic pH values
29. Define Aw, hypotonic, hypertonic, plasmolysis, halophiles, halotolerant, osmophiles.
• Aw – water activity: the ratio of the water vapor pressure of a solution to the water vapor pressure of pure water; measures the availability of water • Hypotonic: solute concentration on the outside of the cell are lower than the cytoplasm
• Hypertonic: solute concentration is greater on the outside of the cell relative to the cytoplasm
• Plasmolysis: loss of water, dehydration of the cytoplasm, shrinkage of the cell membrane away from the cell wall
• Halophiles: require high concentration of sodium chloride to grow • Halotolerant: are capable of growth in moderate concentrations of salt • Osmophiles: are able to grow in environments where sugar concentration are excessive
30. Understand what happens to a cell when it is placed in a hypotonic or hypertonic environment.
• Hypotonic: when placed in a hypotonic environment water will normally diffuse into the cell. However, the cells are not normally harmed because the rigid cell wall that protects the membrane from being damaged by the osmotic pressure exerted against it.
• Hypertonic: in hypertonic conditions, water will diffuse out of the cell. This results in plasmolysis which is dangerous for the cell. The dehydration of the cytoplasm can cause considerable and often irreversible damage to the metabolic machinery of the cell.
31. Know which organism (that we tested) should have been able to grow at the highest salt concentration.
The lab mid-term exam will include the following types of questions: multiple choice, true/false, matching, short answer, fill-in the blank (with word bank), and practical-type questions in which you will have to look at something (such as a slide, plate, tube, etc.) and answer a question about it.