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94 terms

Micro Lab Final- Dr. Moore

HU Microbiology lab final 2014
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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