MICROBIOLOGY LAB PRACTICAL
Identify the purpose of the type of stain is used of a slide (e.g. what it allows you to identify within or about a bacterial cell).
Flagella stain: the purpose of this stain is to determine whether a microorganism has flagella and the location of where it is. acid fast stain: Used to identify bacteria that contain mycolic acids in their cell walls negative stain: used to clearly see the cell morphology and arrangement of the microorganism endospore stain: used to be able to see the bacterial endosopores; can be spherical or elliptical capsule stain: used to distinguish between bacterial cell and capsule of cell
Identify if a bacterial culture is Staphylococcus aureus, another type of Staphylococcus species, or not a Staphylococcus species on Mannitol salt agar
-Staphylococcus aureus produce yellow colonies with yellow zones -Staphylococci produce small pink or red colonies with no colour change to the medium -if it's not a staphylococcus species at all, there will be no growth at all
9. Calculate original cell density of a bacterial culture
OCD= CFU/(Dilution factor x Volume)= CFU/sample volume= CFU/mL
Utilize a DNA ladder to determine the size of DNA fragments visible on an agarose gel.
draw a line from the BOTTOM of the fragment to where it matches up on the ladder.
On an agar plate or image, classify bacterial hemolysis as beta, alpha, or gamma and know the specific types of media used to identify hemolysis.
gamma hemolysis: no change in the medium beta hemolysis: clearing around growth alpha hemolysis: greening around growth
8. Distinguish the results of the Gram positive and Gram negative bacteria, and their cellular envelope differences.
gram positive-stain purple and their cell walls are made up of thick layers of peptidoglycan gram-negative- stain pink and cell walls are made of a thin layer of peptidoglycan; also has an outer membrane that consists of lipopolysaccharides
Identify the type of stain technique and specialized stain used when viewing a slide from the following list: capsule stain, flagella stain, acid fast stain, negative stain, endospore stain
Capsule stain: result: capsule is left unstained as a fat halo between cells and background; negative staining technique; negatively charged acidic dye (congo red) stains background and positively charged basic stain colorizes cell Flagella stain: flagella are thin fragile structures used by microorganisms for motility; stains used for this are mordant and silver nitrate which help set the dye by plumping the flagella so they can be stained and easier to observe acid-fast stain: acid-fast stains: fuschia non acid-fast cells: green or blue primary stain used is carbol fuchsin, which has phenol and helps go through the cell wall. steam is also used to help it penetrate the waxy layer better. then, decolorize the cells and put the counterstain methylene blue on the slide endospore stain: Schaeffer-Fulton method primary stain: malachite green which is forced in my steaming the emulsion negative stain: staining the background of the slide, bacteria look clear in this stain
2. Identify bacterial colony morphology (whole colony shape, margin, and elevation) using appropriate terminology
Colony morphology can be seen with naked eye (macroscopic): SIZE of the colony: helpful, however keep in mind it is relative to growth time of colony SHAPE: -round: circular - irregular: no real shape - filamentous: looks like filaments (spaghetti- like w a center ball) - rhizoid: looks like roots MARGIN: smooth/entire: entire colony has a smooth/round edge irregular/erose: the edge is jagged lobate: the edge is like the lobe of a flower/your ears filamentous: always goes w/ filamentous colony shape rhizoid: the edge looks like tree roots SURFACE: smooth, rough, wrinkly, dull, shiny TEXTURE: moist, mucoid (slimy), butyrous (buttery), dry ELEVATION: flat, raised (uniformly raised all the way across), convex (raised mostly in the middle like a hill), umbonate (a hill on top of a hill), growth into medium (burrows into the medium) COLOR/OPTICAL PROPERTIES: pigment(color), opaque (can't see through it), translucent(can see through it a little bit), transparent (can completely see through it; this is uncommon)
10. Calculate the total/final dilution factor in a dilution tube following a series of dilutions.
DF= DF1 x DF2 x DF3 example= (1/10) x (1/10) x(1/10) = final dilution factor after 3 dilutions is 1/1000
7. Calculate the dilution factor from a single, simple dilution.
DF= The volume of the solute or stock concentrate added/ the total volume in your dilution example: the dilution factor for lemonade is 1 can of frozen lemonade concentrate/ 3 cans of liquid making a 1/3 dilution ("1 in 3 dilution"), and the concentrate is diluted by a factor of 3.
For a given agarose gel electrophoresis set up or image, be able to identify the location of the positive and negative electrodes based on the direction the DNA bands migrated from the loading wells.
DNA fragments are negatively charged, and therefore move towards the positive electrode. The end of the box where the wells is where the negative electrode will be, and the positive electrodes are at the opposite end, where the DNA will migrate towards.
Know which Gram designation EMB agar and Mannitol salt agar select for and against.
EMB AGAR- selective because it inhibits the growth of Gram-positive bacteria and promotes the growth of Gram-negative bacteria; differential because it can distinguish bacteria that can ferment lactose from those that can't. In addition, it can identify vigorous fermentation of lactose. MANNITOL SALT AGAR: used mainly to identify the bacteria Staphylococcus aureus from other Staphylococcus species. It is selective because it inhibits the growth of Gram-negative bacteria, while encouraging the growth of Gram-positive bacteria. It is differential because it can distinguish bacteria that can ferment mannitol from those that can't.
For each of the four hydrolytic tests, match the hydrolytic enzyme examined by the test and the type of molecule the enzyme hydrolyzes
GELATINASE TEST - hydrolytic enzyme gelatinase. Gelatinase is responsible for breaking down the protein gelatin into smaller peptide fragments and amino acids positive for gelatinase: liquification negative for gelatinase: solid AMYLASE TEST (starch agar): hydrolytic enzyme amylase. breaks down the polysaccharide starch into dextrins (smaller polymers), maltose (disaccharide), and some glucose. procedure for test: iodine is added to starch positive for amylase: no starch present; glucose is present and clearing around growth negative for amylase: starch is still present; blue/black/brown CASEASE TEST (Milk Agar): hydrolytic enzyme casease, which breaks down the milk protein casein into smaller peptides and amino acids. procedure: substrate casein is observed for breakdown in this test. positive for casease: clearing around growth negative for casease: white agar (no reaction) UREASE TEST: hydrolytic enzyme urease, which hydrolyzing urea (a nitrogenous waste product) into a usable form of nitrogen for bacteria. Urease hydrolyzes urea into the basic compound ammonia (NH3) and carbon dioxide. procedure: Since ammonia is a basic substance (pH of 12), a pH indicator (phenol red) is used in this test to determine if it's present positive for urease: Fuschia negative for urease: red/orange
Correctly identify blood type by the agglutination reaction: procedure: antibody is added to test for agglutination
If A antigen is positive (clumps), then the person has type A blood If B antigen is positive (clumps), the person has type B blood If both A and B clump, then the person has type AB blood If neither A or B clump, then the person has type O blood
Distinguish between molds and yeast on their characteristics when grown in culture on an agar plate or on a slide.
MOLD: filamentous and multicellular; produce both sexual and asexual reproductive spores. ways they appear on an agar plate/slides: -slides show filaments and sporangium -fuzzy" growth of this mold on the plate and are able to clearly see the different filaments and the conidiophore, where the spores are released from, and the chain of spores extending outward. You can also see the small circular spores that broke off from the conidiophore all throughout the surrounding area. - "fuzzy", hairy, dark, and/or spotted - brown-black growth of the conidia (chains of spores) -may appear powdery (e.g. Penicillium), w/ color ranging from blue-green, green-grey, and white) and may have visible spore formations YEAST: unicellular and non-filamentous; reproduce asexually ways they appear on agar plate/slide: -on slides: appear almost like cocci bacteria, but larger in size and show visible organelles at higher magnifications. -on agar plates: yeast colonies look more similar to bacterial colonies than mold, but is a fungus
5. Describe bacterial cell morphology and arrangement using appropriate terminology (cocci, bacilli, spirilla, single cell, diplo, strepto, staphylo, sarcina, and tetrad).
MORPHOLGY= shape cocci- circle bacilli-rod spirilla- spiral Some variations include spirochetes (flexible spirals), coccobacilli (shorter rods than bacilli), and vibrios (slightly curve-shaped rods) ARRANGEMENT= associations single cell- completely dissociated from each other diplo- : Pairs of cells strepto- : Chains of cells staphylo-: Clumps/clusters of cells tetrad: Groups of four sarcina: Groups of eight
Identify a successful streak for isolation
a streak plate is successful if: -you can see a clear colony -you can pull out one individual colony - all bacteria in an isolated colony are genetically identical (essentially showing they are members of the same species) -you can have 2 different type of bacteria and still have a successful streak plate if you have an isolated colony of each
4. Calculate total magnification
total magnification= ocular X objective example: 10x (ocular lens) X 40x (objective) = 10X40= 400x total magnification
In a given bacterial sample, classify bacteria as coagulase positive or negative.
coagulase positive: agglutination or clumping coagulase negative: no agglutination (may appear cloudy)
Microscopically identify amoeba, paramecia, and euglena.
euglena- locomotion : flagella; nuclei are not visualized as well as other 2 amoeba-locomotion : pseudopodia; ;largest from the 3; darkly stained nuclei visible in the central region paramecium- locomotion :cilia; slipper shape; ;larger than euglena; food vacuoles and nuclei are visible
Identify whether fermentation has occurred and if so what fermentation products are present in a phenol red broth tube.
fermentation has occurred if the red broth turns yellow as this indicates it turned from sugar to acidic. if a bubble forms at the top of the Durham tube, this means gas was also produced. (acid and gas are byproducts of fermentation)
3. Identify whether a bacterial colony is pigmented or not
if it has color it is pigmented i.e. yellow, brown, pink etc. if it is white/ clear then it does not have pigment
11. Indicate the use of micropippettors, how to set them for a specific volume.
micropipettes are used to measure small volumes of liquids accurately in a lab for 100-1000s top number- thousands middle number- hundreds bottom number-tens for 10-100 micropipettes: top number- hundreds middle number- tens bottom number- ones for smaller micropipette ranges i.e. 2-20 uL: top number (tens), middle number (ones),bottom number (tenth place decimal) how to set for a specific volume- set the settings to the number of microliters you want by twisting it until the desired amount and then put the tip of the micropipette on and then draw the liquid up.
In a given slant tube of bacteria, classify the bacteria as catalase positive or negative and indicate the molecule responsible for a positive catalase test.
molecule used for test is hydrogen peroxide (H2O2) catalase positive : a lot of bubbles catalase negative: little/no bubbles
Identify motile and non-motile bacteria in SIM media, using their hallmark characteristics.
motile (Can move/positive): spreading from stab line non-motile (cannot move/negative): no spreading from stab line
Identify bacteria as lactose fermenters and non-lactose fermenters on EMB agar
non-lactose fermenters: poor/ no growth, gram-positive & no color lactose fermenters: good growth pink- gram-negative (possible coliform) black/metallic green/purple- high acid production, gram-negative (probable coliform) colorless: gram-negative (noncoliform) BASICALLY, no color usually indicates its a non-lactose fermenter
1. Recognize the parts of a microscope and the function of each
ocular lens- magnifies the image and it used to view the specimen revolving nose piece- holds objective lenses and is rotated when changing power objective lenses- includes 4x, 10x, 40x, and 100x(used for oil immersion aka bacterial lens) stage- slides are placed here for viewing Iris Diaphragm- Increases/ decreases the amount of light to better view your specimen Lamp/Light- acts as a light source On/off switch- turns light on/off mechanical stage adjustment knob- moves the stage fine adjustment knob- (usually the smaller knob)used to focus higher power objectives coarse adjustment knob- should only be used to focus lower power objectives Stage clip- secures slide in place for viewing arm- used to carry microscope
6.Identify growth patterns in broth using appropriate terminology (uniform fine turbidity, flocculent, sediment and pellicle).
pellicle - growth on top of the broth culture sediment - growth that settles on the bottom of the broth culture tube flocculent - clumps of growth within the broth culture. uniform turbidity - an even opaqueness (cloudiness) to the broth culture
Identify whether or not an organism is able to reduce nitrate to nitrite or some other nitrogenous compound, when viewing results from Phase I (addition of reagents A and B) or Phase II (addition of zinc).
phase 1: positive:if color turns red negative: no color change phase II (add powdered zinc): positive: no change negative: red
Identify amylase positive and negative bacteria, using their hallmark characteristics.
positive for amylase: no starch present; glucose is present and clearing around growth negative for amylase: starch is still present; blue/black/brown
Identify casease positive and negative bacteria, using their hallmark characteristics.
positive for casease: clearing around growth negative for casease: white agar (no reaction)
Identify gelatinase positive and negative bacteria, using their hallmark characteristics.
positive for gelatinase: liquification negative for gelatinase: solid
Identify urease positive and negative bacteria, using their hallmark characteristics.
positive for urease: Fuschia negative for urease: red/orange
Using zone of inhibition measurements and a data table, classify a bacterium's antibiotic susceptibility (susceptible, intermediate or resistant) using the Kirby-Bauer test.
resistant- little to no zone of inhibition intermediate- medium zone of inhibition susceptible- large zone of inhibition compare to chart, measure diameter of clearing
Identify sulfur reduction positive and negative bacteria using SIM media, using their hallmark characteristics.
sulfur reduction: positive-black ------can reduce sulfur negative- no change------cannot reduce sulfur