Lab Test 3 b.board notes

(From Blackboard Lab review material, I think the Final is Thursday July 28th.) 

KEY TO REVIEW SHEET FOR LAB TEST 3

• If you only are looking at my answers, and not trying them yourself, you are cheating yourself out of the real value of the review!!

1-What are the basic differences between exo- and endo-enzymes? (Include location and type of substrate)
Exoenzymes are those which are secreted out of the bacterial cell to act upon substrates within the environment. (Usually these substrates are large macromolecules, which are broken down in to smaller monomers.  This may be seen as a clearing in medium where the substrate is insoluble, thus a suspension, while the end products are soluble and therefore clear.) Examples include: coagulase, hemolysins, lipase, gelatinase, caseinase, amylase.
Endoenzymes are those which are retained within the bacterial cell and utilized for activities such as energy production, DNA replication, and movement. The substrates of these enzymes are smaller molecules, such as sugars, which are found within the bacterial cell.)  Examples include all of the fermentation enzymes, citrase, thiosulfate reductase, etc.

2-What is the one principle upon which all quick (serological) tests work? What are the three types of serological tests, and what is an example of each?
All serological tests involve the specific binding of antigens and the antibodies they elicit like lock and key. If you have either the antigen or the antibody from the patient, you can use known labeled partner components to identify the unknown.
Precipitations involve soluble antigens and antibodies, which appear like a film in agar. (Ouchterlony plate)
Agglutinations occur naturally when cellular antigens bind to antibodies, making a clump visible to the naked eye. Artificial agglutinations involve either the antigen or antibody bound to a latex bead, which clumps with the other partner component. (Staph.ID  latex agglutination, titers, blood typing)
ELISA reactions are color change reactions, in which either the antigen or antibody is bound to a dye which changes color when antigen and antibody bind. (quick pregnancy tests, quick Strep. tests)

3-When any biochemical reaction is going on in the lab, we detect the presence of the enzymes indirectly, for the most part. What do the indirect tests often indicate (two answers), and what is one direct enzyme detection test which we did?
Indirect tests usually indicate the disappearance of the substrate, or the appearance of an end product.
Direct detection of the cytochrome oxidase enzyme was performed on the Dry-slide.

4-Describe the tests / media that you would do to confirm a positive Strep. throat diagnosis. (Be detailed, tell what a positive rxn. would look like for each.)
On a throat culture (blood plate) you would look for many small colonies of beta hemolysis (complete), with a zone of inhibition around the A disc (bacitracin), which often shows up as a red, unlysed circle. Only group A, beta hemolytic Strep. include the pathogen Strep. pyogenes which causes Strep. throat. To confirm the identity of the organism biochemically, you would want to grow it on TSA so that you could do a catalase test to ensure that you indeed had Strep. and not Staph or Micrococcus.
 
5-Describe the tests / media that you would do to confirm a positive Staph. aureus contamination of a food. (Be detailed, tell what a positive rxn. would look like for each)
First you would streak the food sample on a MSA plate which would grow nice sized white colonies with only Staph. If the isolated colonies turn the medium yellow, you would know that they were mannitol fermenters and potentially Staph. aureus. To confirm this, you would inoculate the isolated organism into a rabbit plasma tube to check for the production of coagulase. A solidification of the plasma within 24 hours would be a positive test for Staph. aureus.
 
6-Describe the tests / media that you would do to confirm E. coli from a water or meat sample. (Be detailed, tell what a positive rxn. would look like for each)
First streak the organism on an EMB or MAC plate to select for gram -. If there was growth and lactose fermentation (pink on MAC, purple on EMB), then you would suspect E.coli or Enterobacter. (Note: if you saw a greenish-gold metallic sheen to the growth on EMB, you would strongly suspect E. coli.) Next you would inoculate the IMViC series to differentiate between E.coli and Enterobacter. If the results were + + - - you would confirm that it was indeed E. coli. (This means + for indole - red top zone in tryptone broth after incubation and the addition of Kovak's; + for methyl red - red when grown in a glucose broth and adding methyl red; - for Voges-Proskaur - colorless after growing in a glucose broth and adding VP reagents A and B, then agitating for 10 minutes; and - for Citrate utilization - citrate agar remains green after stab/streak inoculation and incubation)
 
7-Explain the following:

• Why does glucose fermentation show up in the butt of a TSIA tube at 24 hrs.?
• Because the small amount of glucose will have been used up at around 12 hours, leaving the organism to respire the sugars and proteins at the surface. The surface will become alkaline by 24 hours, but the butt will remain yellow, since no respiration can go on in the anaerobic depths of the stab.
•  
• What is the actual black substance formed in a positive H₂S test, and why is it only seen under the surface of agar?
• The black substance is the indicator FeS, produced chemically when the organism produces H₂S. This cannot be seen on the surface, because the H₂S has to percolate up through the media to encounter the Fe and combine with it to make FeS.
•  
• Why do you never put a P disc on a throat culture for Strep.?
• The P disc contains optichin, a drug that Strep. pneumoniae is sensitive to. This is used to detect the presence of Strep. pneumo., but will never be helpful in a throat culture, since this organism never causes Strep. throat.
•  
• Why should you never see a bubble in a Durham tube if the color of the medium is purple?
• Gases such as CO₂ and H₂ are made as possible end products of fermentation. If the tube's broth stays purple, there is no fermentation occurring, so no gases would be made. (Respiration does not produce significant amounts of gas during normal incubation periods.)
•   
• Why is there agar in SIM?
• The small amount of agar prevents organisms from settling or rising in the medium, therefore trapping them in the location in which they were placed during the stab. This allows motility to be observed as the ONLY method that organisms could use to spred throughout the medium. Full agar amount = false negative as they are trapped in place.  No agar = false positive as diffusion disperses them throughout the medium.
•  
• Why must the MR and VP reactions always give opposite reactions if one is positive?
• Since the MR and VP tests are testing for different end products from the same substrate (glucose fermentation), there could not be both reactions present in the same organism. Bacteria use only one pathway, either as mixed acid fermenters (such as E.coli) to produce strong acids and a positive MR;  or as butanediol fermenters (such as Enterobacter), to give a positive VP.

8-How does a selective medium differ from a differential medium? Why would you choose to use this type of medium, instead of one which allows everything to grow?
A selective medium inhibits the growth of some organisms while encouraging the growth of others. This is most useful is you are looking for one specific type of organism in a mixed culture. Using a selective medium will eliminate many of the unwanted organisms from consideration, saving you time, work, and medium (therefore money).

9-Complete the following chart of biochemical reactions:

SUBSTRATE	ENZYME	END PRODUCT	INDIC. OF + TEST
lipids	lipase	glycerol and fatty acids	clear around streak
red blood cells and hemoglobin	Beta hemolysin	Fe and amino acids	clear, colorless blood plate around growth
citrate	citrase	pyruvate and CO2	blue color due to Na causing alkalinity
starch	amylase, maltase	glucose	clear after iodine
H2O2	catalase	H2O + O2 gas	bubbles after H2O2 added
tryptophan	tryptophanase	indole + pyruvate	red with Kovak’s
cysteine thiosulfate	cysteine desulfurase thiosulfate reductase	H2S + pyruvate +NH3	black precipitate (FeS) with Fe in medium
fibrinogen	coagulase	fibrin	plasma solidifies
casein	caseinase	amino acids	clear around growth

10-Give the ingredient(s) which allow the media below to have the property listed in parentheses:

• MAC (selective) crystal violet and bile salts
• MSA (differential) mannitol and pH indicator
• blood (differential) red blood cells
• PEA (selective) alcohol
• EMB (differential) eosin and methylene blue

11-Complete the following statements:

• If it's green/gold metallic on an EMB plate, it must be... E.coli  (and Gram - also)
• If it grows well on a PEA plate, it must be... a hardy gram + cocci (not Strep.)
• If it grows well with white colonies on a MSA plate, it must be... Staph.
• If it grows well on a MAC plate, it must be... gram negative
• If there is yellow agar on a MSA plate it must be... a mannitol fermenter (likely Staph. aureus)
• If it is colorless on an EMB plate, it must be... a lactose non-fermenter (and Gram –  also)

12-Why do you always do the IMViC tests together, and why must you record them in order?

All four tests are opposite for E. coli and Enterobacter, and therefore all four must be done to confirm the identity of an unknown lactose fermenting gram - rod. E.coli is + + - - , while Enterobacter is - - + +. They must be recorded in order (Indole, Methyl Red, Voges-Proskaur, Citrate) to use the above shorthand.

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 Lab test 3 spring 2012

1.       Complete the following chart of biochemical reactions studied in lab. (@30pts.)

SUBSTRATE	ENZYME	END PRODUCT(S)	INDICAT. OF + TEST
	caseinase
		strong acids (pH = 4.0)	Color= Reagent(s) added=
thiosulfate (S2O3)			Color = Molecule formed=
	α (alpha) hemolysin
		pyruvate, NH3, indole	Color= Reagent added=
			blue color and growth
fibrinogen
		acids and CO2 gas	broth color= Gas in                     tube
			yellow color on MSA plate
	gelatinase

APPLICATIONS:  Use your knowledge of biochemical and serological tests to answer the following.

2.       Elizabeth's lab group was assigned with making up the media for their lab section, but they had some problems. Briefly tell what reaction would be undetectable if each media below was prepared incorrectly as indicated below: @10 pts.

• No agar in the SIM medium-

• More glucose than lactose and sucrose in the TSIA medium-

• No salt in the MSA plates-

• No pH indicator in the MAC plates-

• No glucose available for any of the tubes in the IMViC series- (hint: two of these)

3.       Morgan is a nurse practitioner in a rural clinic where she must do her own tests and cultures.  She suspects that a patient has Strep. throat.  @16pts.

a)       .                                               - What plate medium will she use to grow the throat sample?

b)      .                                               - What biochemical reaction can be read on the medium in a)?

c)       .                                               - Is the enzyme demonstrated in reaction  b) an endoenzyme or an exoenzyme?

d)      .                                               - What would the catalase reaction of this  Strep. pathogen be, once isolated?

e)      .                                               - colonies  showing sensitivity to what drug would confirm strep. throat?

f)        .                                               - what species of Streptococcus causes strep throat?

g)       .__________________-To which Lancefield group does this species of Strep belong?

h)      .__________________-A “quick” Strep test for this species would be which of the 3 types of serological tests?

4.       Completion/Fill in the Blank:  For each statement below, give the missing word or words  in the box to the left of the statement:  @10pts.

 

·         To detect a biochemical reaction, we rarely test for the presence of the enzyme directly; instead we indirectly detect either ? or ?

 

·         The substrates of exoenzymes are all alike in that they are ?

 

·         Two plate media which are selective for gram negatives are ?

 

·         The most definitive test to identify Staphylococcus aureus is the ?

 

·         All serological reactions involve binding of  ? to ?

PRACTICAL QUESTIONS:  For each of the following, view the set-up and answer the question(s) specific to each.

4pts. 5-Record ALL of the reactions of this organism, seen here in TSIA medium.  (Indicate if this organism is positive or negative for each reaction.)

2pts. 6-What can be determined about the organism growing on this plate?

3pts. 7-What type of hemolysis is seen here?

Is this organism likely to be a pathogen?

2pts. 8-What can be determined about this organism’s fermentative abilities?

3pts. 9-Does organism A have the ability to digest casein?

What substance(s) are present in the clear area of the plate at the arrow?

2pts. 11-Is the organism seen here likely to be aerobic or anaerobic?  What is this reaction?

3pts. 12-List ALL of the things can you determine about this organism, based on its growth here.

5pts. 13-Record the results IMViC reactions seen here.  (Reagents have been added where needed.)

What is the likely genus of this organism?

4pts. 14-What reactions can you determine about this organism, seen here in SIM medium?

                What third reaction could be recorded here, if you added another reagent?  (What

                reagent would you need?)

2pts. 15-What information can you determine about the organism which was inoculated onto this plate, and

                incubated at optimal conditions?

2pts. 16-Which of the three basic types of serological reactions is this?  To what item(s) is this person allergic?