Micro Lab Final- Dr. Moore

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HU Microbiology lab final 2014

Colony

progeny derived from a single cell with identical genetic characteristics. clump of growth on agar

CFUs/ Colony forming units

bacteria arranged in groups

Streak Plate

to isolate bacteria; concentration gradient decreasing bacterial quantity across the plate

Loop Dilution

most common method for the quantitation of bacteria from specimens. A loop holds 1-10 micro liters of sample and spread it evenly on agar. It can determine the number of organisms in the original sample.

What is the difference between qualitative and quantitative methods of colony isolation (streak plate versus loop plate)

Qualitative (used mainly in streak plates) defines the quality of the test/organism. It is able to isolate a colony by decreasing bacterial quantity across the agar plate. Quantitative (used more in loop dilution and pour plates) determines the clinical/industrial count of the organism.

Why is the streak plate important in obtaining a pure culture?

It can separate organisms for determination of purity and colony morphology. It "thins out" a heavy population of bacteria to isolate a colony

Monosaccharides

glucose, fructose, mannose, mannitol, inositol

Disacchrides

sucrose, lactose, maltose

polysacchrides

starch, glycogen, dextrans, agar

Durham tube

inverted tube to determine fermentation with the production of gas

OF test

oxidative-fermentative tests for the fermentation of glucose

What are some of the different sugars that may be tested using the carbohydrate fermentation tests

fructose, galactose, mannose, ribose, arabinose, xylose, ribulose, sorbitol, inositol, mannitol, glycerol, sucrose, lactose, maltose, starch, glycogen, agar

what is the most common pH indicator used for fermentation studies?

Bromcresol purple

What are the 3 possible interpretations for the O-F test?

Fermentative- Both tubes turn yellow
Oxidative: only the uncovered test turns yellow, the other stays green. Metabolism of glucose is dependent on the presence of oxygen
Non-saccharolytic: both tubes stay green with no color change. indicates that the carbohydrate cannot be broken down

What does an Orange MR test mean?

orange counts as negative indicating lesser amounts of acid production

Why is the catalase test important?

Whether or not the organism can convert H202 to nontoxic components. Useful in the identification of organisms

What are cytochromes? what cytochrome is found in the oxidase test?

Cytochromes are iron containing hemoproteins that act as the last link in the electron transport chain of aerobic respiration by transferring electrons to oxygen with the formation of water and are usually present only in aerobic or facultative anaerobic organisms. Cytochrome C is found in the the oxidase test

What does B-gal do in the ONPG test?

it cleaves lactose analogue (ONPG) into galactose and a yellow nitrophenolate ion

Describe the importance of adding Zinc to a clear nitrate tube

Confirms a true negative reaction; the test only detects nitrites and any further processing of the nitrite leads to a false negative. If Zinc is added to the tube and it stays clear then it means the nitrates were reduced to products other than nitrites like ammonia or N2. If it turns red then it means the organisms did not reduce the nitrates

Is it possible to have gas production in a durham tube fermentation without acid production?

...

What would happen if an organism used up all the carbohydrates in a fermentation tube? What would it use for energy?

It will start to break down amino acids. Eventually die when everything in the tube is used up

Would you expect most anaerobes to be oxidase positive? Why or why not?

can be, but most likely not because they do not use aerobic respiration

If you had a nitrate test which was colorless after adding zinc what would the reaction be and why did you not see the reaction before adding zinc?

This would mean that the nitrates had been reduced to other products other that nitrites, such as ammonia or N2. Zinc ions reduce nitrates to nitrites. This test result is still positive for nitrate reduction

When Pseudomonas aeruginosa is inoculated into a glucose O-F tube it breaks down the glucose oxidatively into acids and other byproducts. But when Pseudomonas is inoculated into a glucose purple broth no change in pH is indicated. Why??

The pH range for bromcresol purple (dye used in the glucose purple broth) is 5.4 to 7.0. The pH range for Bromothymol blue used in the O-F tube test is 6.0-7.6. Pseudomonas aeruginosa would not have been able to produce a large enough amount of acids to change the color of the indicator for the glucose purple broth.

Random shotgun sequencing

technique in which small pieces of a genome are sequenced and the sequences are then assembled by a computer; sped up the Human Genome Project process

bioinformatices

study of gene sequence and function through computer assisted analysis

GenBank

web based database that stores complete and partial DNA sequences

Polymerase Chain Reaction

exponential amplification of a defined DNA sequence using sequence specific primers

Electrophoresis

techinique to sort and separate DNA fragments based on size. Placed on agarose gel and treated with ethidium bromide which fluoresces on exposure to UV light

List the steps of determining a nucleotide sequence from an organism

1. Acquire sufficient amount of DNA for analysis; PCR
2. Electrophoresis to separate DNA by size
3. Cut DNA from gel and removed from agarose matrix
4. Amplified DNA + a single primer sent to refernece lab to sequences the DNA using the chain termination method
5. BLAST analysis

Explain the importance of GenBank and bioinformatics

GenBank acquires information that is instrumental in correlating the virulence genes of unrelated pathogens, exploring new/reemerging pathogens as well as developing screening methods for various human diseases
Bioinformatic analysis can convert DNA to RNA to study RNA function or Protein function within cells

What does BLAST stand for?

Basic Local alignment Search Tool

Define score and E value

Score is a measure of the similarity of the query to the sequence shown. E value is the probability due to chance that there is another alignment with a similarity greater than the given S score.

If you have Ns in your sequence what does that mean?

the base pair is indeterminable and the N is the placeholder

Transformation

The uptake of naked DNA

Ori Site

binds DNA replication initiation factors

bla

gene that encodes for Beta lactamase which is involved in penicillin resistance

arabinose

5 carbon sugar

Green fluorescent protein

encoded for by pGLO gene from jelly fish aequorea Victoria

Why are operons important?

Groups of related genes that are often clustered together and transcribed into RNA from on promoter. Operons produce enzymes or protein products that break down arabinose and turn araC to its original shape which turns off transcription

Where is GFP found in nature and what does GFP stand for?

GFP comes from the Jellyfish aequorea victoria. Stands for Green fluorescent protein

What is the function of the araC complex?

The araC is a DNA binding protein initiates transcription by changing shape and binding RNA polymerase which in turn transcribes the structural genes araA, araB, and araD. When the structural genes are replaced with GFP it transcribes GFP

Virulent

infect permissive bacteria, replicate within the host cell shutting down host cell replication and ultimately lysing the host cell

Permissive bacteria

Bacteria that allows for virulent phages to hijack host cell's replicases and transcriptases

Lytic cycle

life cycle of phage replication

temperate phages

infect susceptible host cells but their nucelic acid incorporates into the host cell genome

prophage

phage genome

lysogenic cycle

prophage can be eliminated from the bacterial DNA and re-enter the lytic cycle

phage conversion

phages that encode for toxins which impart virulence to the organism

plaques:

clearing produced by virulent phages. They lyse target cells and make plaques

Trypticase soy agar

all purpose medium supports the growth of most bct culture.

Selective medium

inhibits the growth of some microbes, promotes the growth of others

MSA

staph will only grow and inhibits others

MacConkeys

differential medium and selective medium. It can determine lactose fermentiation, pink colonies = ferment lactose. colorless = non-lactose fermenters. It differentiates one organism from another

Enrichment medium

nutrients that favor growth of certain organisms

selenite broth

enriches members of the salmonella group so taht they multiply faster then other fecal organisms and outnumber their competitors

non-defined

used in micro lab, exact chemical composition is unknown

chemically defined medium

exact composition is known as well as the exact concentration of every chemical

Why is macConkey agar both selective and differential?

It is both selective and differential becuase it can have bile salt added which inhibits gram + growth and c Cokeys contains lactose and a pH indicator to differentiate one organism from another by looking at the color

What is the basic all purpose media used in labs?

trypticase Soy agar (TSA)

What is the function of a decarboxylase?

substrate specific enzyme capable of attacking the carboxyl group of amino acid, forming alkaline - reacting amines which forms carbon dioxide as a second product

What is the purpose of the control tube in a decarboxylase test?

verifies acidic conditions were obtained during fermentation

Describe the positive and negative reactions for a decarboxylase test? what are the 3 different amino acids taht we use for this test?

positve is purple to yellow to purple. Lysine, Ornithine, Arginine

Describe the reactions for the phenylalanine deaminase and indole tests. Remember to include reagents used.

Phenylalanine test: Phenylalanine is an amino acid that upon deamination forms a keto acid, phenylpyruvic acid. The test depends on the detection of the acid and is positive if after incubation, a green color develops after the addition of 10% ferric acid. Indole test: Part of sim test, add 10 drops of Kovac's reagent, a positive result is red at the top. Negative is yellow at the top. Positive means that Tryptophan was metabolized.
6. page 103 question 2,

Describe the gelatin and starch hydrolysis

Gelatin hydrolysis is determined by the medium's inability to solidify on refrigeration because it has been broken down into amino acids. Starch hydrolysis can be determined by the clearing zone around the hydrolyzed region of bacterial growth after addition of lugol's iodine. Starch hydrolysis if positive will have the starch degraded into individual carbohydrate moieties

Esculin hydrolysis

esculin is hydrolyzed into esculetin which reacts with ferric citrate. Ferric citrate is a part of the medium and turns a dark brown color showing positive for esculing hydrolysis

Urease

positive is pink which shows the breakdown of urea into NH3, tests for the enzyme urease

Coliforms

gram - rod shaped bct. normally present in the intestine

permeases

membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy

simmons citrate agar

used to determine if citrate is the sole carbon source, contains mineral salts, citrate, and ammonium ions

beta-hemolysis

a clear zone surrounding the colony after lysing erythrocytes.

alpha-hemolysis

a green zone surrounding the bacterial colony afterthe lusis of erythrocytes

gamma-hemolytic

non-hemolytic colonies

Enterotoxins

cause physical changes in the intestinal epithelium

oral fecal route

usually how diarrheal diseases are spread. Individuals eat food or water taht has been contaminated by animal feces containing pahogen

infectious dose

the amount of pathogen required to cause an infection in the host

What is the major pathophysiologic effect of a GI pathogen and how is it treated?

diarrhea which causes dehydration and electrolyte loss. Treated with simple solutions of glucose, salts, and water given orally

List the 4 general methods for the identification of infections

1. macroscopic and/or microscopic identification
2. culture and biochemical characterization
3. Serological analysis
4. molecular analysis

Give some general rules to follow when working with an unknown infectious agent

treat all cultures as pathogenic. use aseptic technique

List some products used clinically for rapid detection of a pathogen

apl 20E, Microscan, and Vitek systesm

culture and sensitivity (c/s)

an antimicrobic sensitivity profile used to determine the therapeutic options for a patient

Kirby-Bauer method

an antimicrobic sensitivity testing using a seeded agar plate with antimicrobic impregnated disks placed on it, the antimicrobic disks form zones of inhibition

minimum inhibitory concentration (MIC)

the least concentrated amount of antimicrobial that completely inhibits growth of the pathogen; inhibits the replication process of bacteria and is used to treat someone with a strong immune system

minimum inhibitory bacteriocidal concentration (MBC)

the minimum antimicrobial concentration capable of killing the pathogen; used to treat someone with a weak immune system such as elderly people, pregnant women, infants and people with HIV

list 3 examples of when to use MBC values of doses for patients

When the patient is elderly, pregnant, has HIV or is an infant

what are the 3 possible determinations from the disk diffusion test?

susceptible, intermediate, resistant

List the procedures used clinically to determine a pathogen based on antigen-antibody reactions

precipitation, agglutination, immunofluorescnece

describe precipitation

the soluble antigen is precipitated by an antibody to form and insoluble complex. This makes it possible to distinguish between separate antigen-antibody reactions produced by different antibody populations present in a serum sample

describe agglutination

the aggregation of antibodies with suspended cells or like-sized particles into clumps that settle. Various bacterial and viral-specific antibodies can be detected by agglutination techniques or variations thereof

describe immunofluorescence

A technique used to identify particular antigens microscopically in tissues or on cells by the binding of a fluorescent antibody conjugate

ELISA

Used to diagnose infections such as Strep throat, flu, c. diff. giardia, and HIV

Describe Latex agglutination in Detail

It is serological test. IN a positive result, the anitbodies bind to the target antigen cells and visual clumps arise. IN a negative result, the antibody binding can't occur because the specific target antigen is not present

Give examples where immunofluorescence tests are helpful

to detect virus cells such as Human B-Lymphotropic virus HBLV) only stain what you want and the background stays dark

What does ELISA stand for

enzyme-linked immunosorbent assay

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