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MICROBIOLOGY EXAM > PRA. PART METHODS (all Q) > Flashcards

PRA. PART METHODS (all Q) Flashcards

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1
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

What is the proceedure of gram staining?

A
  1. Crystal violet, 3 min
  2. Lugol solution,2 min
  3. Wash with alkohol for approx. 20 seconds
  4. Rinse with water
  5. Carbol fuschin, 1 min
  6. Rinse, dry (can use filter paper) and view in microscope with immersion oil on
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2
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

Who developed gram-staining and when?

A

Hans Christian Gram in 1884

he was a Danish doctor and bacteriologist

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3
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

What is gram staining used for?

A

Used to differenziate between G+ and G- bacteria. Crystal violet stains the cell wall (peptidoglycan layer) so G- gets a pink color, while G+ gets a purple/blue color.

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4
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

What is the staining used on endospores? Name two spore forming bacteria that needs this staining.

A

Wirtz-Conclin staining.

Used on BACILLUS and CLOSTRIDIUM

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5
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

Procedure of Wirts-Conclin staining:

A
  1. Heat fixate bacteria and 0,9 % NaCl on the slide
  2. Put 5 % malachite green on slide, heat the slide so it’s steaming (not boiling) –> malachite green penetrates endospores and stain them
  3. Repeat this step 2 times
  4. Wash with water
  5. Stain with carbol fuchsin for 30 seconds –> stains other parts of bacteria red
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6
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

What does stained endospores look like in the microscope?

A

Endospores = green

Other bacteria parts= red

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7
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

Name of common acid-fast staining and what bacteria is it used on?

A

Ziehl-Neelsen staining, used on mycobacterium (e.g. m. tuberculosis, m. bovis, m. marinum)

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8
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

Proceedure of Ziehl-Neelsen staining?

A
  1. Slide fixation by heating
  2. Concentrated Carbolfuschin 3 x 5 minutes heating
  3. Decolour by acid alcohol
  4. Stain by malachite green/methylene blue 20 seconds
  5. Wash with water
  6. Dry the slide - microscope - immersion oil
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9
Q

(Q1) Microscopy of stained slides (gram staining, staining of bacterial endospores, acid-fast staining)

Why does Mycobaterium stain good by Ziehl-Neelson? What does it look like after staining?

A
  • Mycobacteria contain large amounts of lipid substances –> mycolic acids in their cell walls.
  • Acid fast bacilli or slightly curved rods will be bright red after staining.
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10
Q

(Q3) Fluorescent test

Is it quantitative or qualitative?

A

Qualitative

–> only positive or negative reaction.

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11
Q

(Q3) Fluorescent test

Is it direct or indirect?

A

Can be both.

“Direct immunofluorescence uses a fluorophore-conjugated antibody to stain the target protein. Indirect immunofluorescence involves first binding the primary antibody to the target, then detecting the primary antibody using a conjugated secondary antibody.

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12
Q

(Q3) Fluorescent test

Give one example of direct and one for indirect testing:

A
  1. Direct used on Lyssa virus (Rabies)

2. Indirect used on Lawsonia intracellularis

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13
Q

(Q3) Fluorescent test

What is used for visualization?

A

Florescent microscope

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14
Q

(Q3) Fluorescent test

What viruses (causing repiratory infection) is susceptible to diagnosis by immunofluorescence? Name 3.

A
  1. Paramyxoviruses
  2. Orthomyxoviruses
  3. Adenoviruses
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15
Q

(Q3) Fluorescent test

What bacteria can be visualized by UV light, and what special medium is used?

A

Bacteria P. aeruginosa and P. Fluorescens if put on Cetrimide agar.

  • Cetrimide agar = agar for production/detection of pigments only for pseudomonas
  • P. Aeruginosa & P. Fluorescens produce typical biproducts of pigments (pyoverdine, pyocyanin)
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16
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

Give 3 examples of commersial multitest:

A
  • Staphytest for genus Staphylococcus
  • Enterotest for genus Enterobacteriaceae
  • Streptotest for genus Streptococcus
  • Anaerotest for Anaerobic bacteria

= genus must be known before testing

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17
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

What is multitests based on? Does it identify spesific bacteria or groups?

A

Enzymatic activity of bacteria. Identifies groups of bacteria.

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18
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

What is proceedure of multitest?

A

Pure culture is needed.

  1. Measure opacity (by Mc Farlands scale) to right amount of CFU (colony forming units). It must be the same in all bacterial samples.
  2. Put into wells
  3. Incubate at 37 C
  4. Add reagents e.g. parafin
  5. Incubate again
  6. After, evaluate dolor changes and read procedure according to (+) or (-) reaction
    - -> find out species in diagnostic catalogue
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19
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

Give procedure spesific for enterotest:

A

Procedure:

  1. Collect colony & put into liquid form - physiological solution to 3° McFarland Opacity
  2. Measure McFarland opacity in machine (densimeter)
  3. Add 100µl to each well
  4. Add paraffin-oil (anaerobic atmosphere) into 4 wells
  5. Incubation in thermostat for 4 hours at 37℃
  6. Add reagents - 30 min incubation at 37℃
  7. Read colour changes in table
  8. Obtain code of bacterial species
  9. Find out in diagnostic catalogue
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20
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

What is OF-test for and is it used on G+ or G- bacteria?

A

Used to determine if bacteria metabolise carbohydrates (glucose) oxidatively, fermentatively or if it’s non saccharolytic (no ability to use carbohydrates in media).
Used on G- bacteria.

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21
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

What agar is used in OF test and what does it consist of?

A

Semisolid agar, that consist of glucose and bromothymol blue that indicated pH level.
2 tubes are used for 1 bacteria: 1 w/ parafin, 1 without.

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22
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

OF test: If both tubes w/ parafin and without changes color, what is are the bacteria?

A

Facultative anaerobic bacteria

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23
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

TSI Agar stands for?

A

Triple Sugar Iron/ Hain’s test

24
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

TSI test tube contains?

A 
o	Agar
o	pH sensitive dye (phenol red)
o	1% lactose
o	1% sucrose
o	0.1% glucose
o	Sodium thiosulfate 
o	Ferrous sulphate or ferrous ammonium sulphate
25
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

TSI is used for bacteria… ? Give 2 examples:

A

Often ffor enteric bacteria e.g. Salmonella and Shigella.

26
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

TSI results:

A

o Bacteria that ferment any of the 3 sugars produce by-products  usually acids
o Acid changes the colour of phenol red to yellow
o Sugar fermenting e.g. Enterobacteria
o The hydrogen sulphide produced by some bacteria reacts with ferrous sulphate forming ferrous sulphide
o Ferrous sulphide is visible as black precipitate
o The blackening of medium is visible usually in the bottom of the medium
o Sulphide producing bacteria e.g. Salmonella, Proteus, Citrobacter, Edwardsiella

27
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

Oxidase test
Remember examples.

A

• By indirect biological oxidation: emission of oxidase enzyme
o By use of strip test, touch colony with strip & wait for ≅ 1 min  turns blue/dark colour if it produces oxidase (+)
• Oxidase (+) – e.g. pseudomonas, Pasteurella, ornithobacterium (usually aerobic/facultative anaerobic)
• Oxidase (-) – e.g. Enterobacteriaceae (no colour change)

28
Q

(Q4) Biochemical methods (commersial multitest, OF test, TSI agar, oxidase test, catalase test)

Catalase test
Remember examples

A
  • The enzyme, catalase, is produced by bacteria that require using oxygen & protects them from the toxic by-products of oxygen metabolism
  • The destruction of toxic H_2 O_2 by enzyme catalase into H_2 O& O_2 –> observable by presence of bubbles
    • Put drop of H_2 O_2 on slide, touch colony with it – wait for bubbles (+)

-Catalase (+) – e.g. staphylococcus, bacillus, Escherichia, salmonella (bubbles)
Strict aerobes & facultative anaerobes

  • Catalase (-) – e.g. streptococcus, clostridium, ornithobacterium (no bubbles)
    . Anaerobes or facultative anaerobes that only ferment & don’t respire using oxygen as a terminal electron acceptor (e.g. Streptococci)
29
Q

(Q5) CAMP test

Examples of bacteria we use for this test:

A

Used for Str. Agalactiae!

Also, others e.g. Rhodococcus Equi, Actinobacillus Pleuropneumoniae, Streptococcus Agalactiae, Listeria

30
Q

(Q5) CAMP test

What strains are used for the test?

A

S. Aureus and unknown streptococci strain

31
Q

(Q5) CAMP test

Proceedure and result:

A
  1. Make a single line of beta-haemolysin producing S. Aureus down the centre of blood agar plate
    –> Blood agar = (usually) trypticase soy agar + 5% sheep blood
  2. Inoculate a line of beta-haemolytic streptococcus to be identified perpendicular to the staphylococcal line (not touching)
  3. Aerobic incubation at 35°C for 18-24 hours
  4. After incubation period:
    • if haemolysis appears, from alpha to beta  Str. Agalactiae BUT if no haemolysis, then not sure what it is
    • Normal Str. Agalactiae only has mild haemolysis
32
Q

(Q&) Decapsulate test

Everyting about it:

A

Decapsulate test
• Based on cultivation on blood agar with 2 strains together
o 1st strain = S. Aureus in the middle (like CAMP test)
o 2nd strain = P. Multocida Serotype A, is cultivated near the 1st strain
• Staphylococcus produce enzyme, hyaluronidase, which destroy the hyaline capsula of the 2nd strain
• Can then see that line is missing in some part
o If 1cm disappears near S. Aureus, the 2nd strain have capsula
 Strain with capsula is mucotic
o If nothing disappears & there is still a whole line that almost touches the line of the 1st strain, then there’s no capsula
 Strain without capsula is not mucotic
• Advantage
o The whole procedure is fast, carried out in a simple way & its result is evident
o Compared with other currently used tests, this one is substantially cheaper
• Disadvantage
o Reading test results only after 18 hours

33
Q

(Q7) PCR, real-time PCR

What does PCR stand for?
Who invented it and when did he/she get an nobel prize for it?

A

Polymerase chain reaction

Invented by Prof. Kary Mullis which got a Nobel Prize in 1993 for it.

34
Q

(Q7) PCR, real-time PCR

Components of PCR (master mix)

A
  1. THE PAIR OF DNA PRIMERS – oligonukleotide which has specific sequence of nukleotides (20-25)
  2. DNA POLYMERASE (taq) thermoresistant enzyme from Thermus aquaticus (bacteria lives in the exthreme condition in the depth of the sea volcano by the temperature 95°C.
  3. FREE NUCLEOTIDES (adenin, thymin, cytosin, quanin)
  4. BUFFER Complete (Mg+)
  5. WATER for PCR + DNA template!! –> must know
35
Q

(Q7) PCR, real-time PCR

Procedure of PCR:

A
  1. DENATURATION (more than 90°C, 1 min)-separated of DNA molecules
  2. ANNEALING (55-65°C, less then 1 min.) –binding of the primers
  3. EXTENSION (72°C, 30s-2 min)- elongation of template DNA by the nucleotides and complete of the strand.
36
Q

(Q7) PCR, real-time PCR
3 advantages:
3 disadvantages:

A

Advantages:

  1. High sensitivity (from two strands of NA = 107 milion copies DNA)
  2. High specifity (DNA is unique for each organism)
  3. Suitable for detection of viruses, mycobacterias and other organisms in which cultivation isn´t possible or the incubation time is very long.
  4. It isn´t time consuming

Disadvantages:

  1. False positive results (contamination, incorect binding of primers )
  2. False negative results ( autolysis of DNA by UV, freeze)
  3. Expensive methods
37
Q

(Q7) PCR, real-time PCR

All about qPCR:

A
  • Allow quantificate the template of NA isolated from cells, organs, tissues, and body fluits (blood, urine…)
  • Using of probe- flourofor SYBR-GREEN I, which is bind on the doublestrand fragment of DNA
  • Visualisation is on the monitor of PC, we can see the curves and read the amount (quantity) of the amplicon (DNA) in individual cycle.
38
Q

(Q8) MALDI TOF

What does MALDI TOF stand for?

A 
Matrix assisted laser disorption ionization time of flight
MASS SPECTOMETER (imp to know i følge Dana)
39
Q

(Q8) MALDI TOF

What can maldi tof be used for?
Price for maldi tof in CZK?

A

Used for fungi, yeast and bacteria. (the most imp for bacteria ifølge Dana)

Costs 5.000.000 CZK

40
Q

(Q8) MALDI TOF

Describe procedure:

A
  1. Need pure culture
  2. Transfer colonies into each well on plate (about 90) with tooth picker.
  3. After some time (aprox. 15 min), results will show and you can compare with database.
  4. After measuring you obtain the MASS SCORE (imp to know). and it should be 2 or above. If not –> second testing is done.
41
Q

(Q9) Disc diffusion method, broth dilution method

Which is quantitive and which one is qualitative?

A

Disc diffusion method is qualitative, broth dilution method is quantitative

42
Q

(Q9) Disc diffusion method, broth dilution method

What value is important to obatin and what does it stand for?

A

MIC value = minimum inhibitory concentration

43
Q

(Q9) Disc diffusion method, broth dilution method

What documents are used for reference values?

A

NCCLS or EUCAST

44
Q

(Q9) Disc diffusion method, broth dilution method

What is the 3 different results we obtain for a spesific bacteria?

A

Sensitive, intermediary or resistant

45
Q

(Q9) Disc diffusion method, broth dilution method

What agars are used of disc diffusion method, and give 1-2 examples of bacteria using the agar.

A
  1. Mueller hinton = For most common bacteria
  2. Mueller hinton blood agar = for streptococcus and pasteurella
  3. Choclate agar = for actinobacillus, haemophilus and campylobacter
46
Q

(Q9) Disc diffusion method, broth dilution method

Proceedure:

A
  1. Pure culture is used to make suspension (physiological solution and bacteria) with 0,5 degrees McFarland opacity. Then it is diluted 100 times which gives 1.000.000 CFU.
  2. Put on agar
  3. Put concentrated discs with AtB (usually like 6 discs)
47
Q

(Q9) Disc diffusion method, broth dilution method

For broth dilution method procedure:

A
  1. Microtitration plate
  2. Dilution of atb (10-15 different concentrations)
  3. Use bacterial suspension (opacity 1.000.000 CFU)
    Apllicate into well, incubate in 37 degrees.
  4. Result: First ceoncentration of atb which prevent growing of bacteria = MIC
48
Q

(Q10) Tissue culture, cythopathic effect

What is cythopathic effect?

A

Destruction of monolayer

  • Structural changes in a host cell resulting from a viral infection
  • Occurs when the infecting virus cause lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce
49
Q

(Q11 a) Serological methods: ELISA

Procedure:

A
  • Use microtitartion plate (96 wells)
  • Use spesific antigen (usually commersial) to cover each well and wait maybe 1 day so it’s stuck to the walls
  • Second day we add tested serum samples and let incubate maybe 1 hour.
  • Then wash it by washing buffers, usually 3 times.
  • Then add a spesific conjugate (usually commersial antibody (from animal we are testing) which is conjugated with the enzyme horse-radish peroxydase)
  • Incubate 1 hour, during this antibodies bind on antibodies and transfer the enzyme on the reaction.
  • Then wash 3 times
  • Add substrate with chromogen (color), applicate for only 15 min. Usually yellow!
  • Stop it by STOP solution so color stops developing.
  • See result if it’s colors or not. So if color = positive, if no color = negative.

Evaluate color by eyes, or more commonly by ELISA reader.

50
Q

(Q11 c) Immunoblot

Procedure

A

Detection of virus,

  • Usually have some main protein from the virus wich we put on to the gel –> POLYACRYLE AMIDE GEL, and spread the virus by vertical electrophoresis. So proteins are separated.
  • Then proteins are blotted into a nitrocellulose membrane at 110 V for 1 hour - 1,5 hour.
  • After this the nitrocellulose membrane stick on the gel, soak the antigen inside the nit. cell. membrane, and after blotting procedure we cut the membrane (which is a piece of paper 10x6 cm in size ca.)
  • We cut it on special strips and put into chamber and incubate into serum samples.
  • We must have a positive and negative serum test (control tests) , so we can compare with positive samples.
  • After incubation with positive, negative and samples for testing we add conjugate.
  • After incubation and washing we put the substrate.

This test is very demanding.

51
Q

(Q11 e)
Rapid immunochromatographic test/ SNAP test:

Procedure:

A

Popular test.
Fast test from blood samples. LARGE ADVANTAGE
Positive result = two red stripes in the evaluating window. 1 red strip = negative. NO STRIP = test is not valid.
We have results after 15- 20 min. Often used on FeLV and FIV. The test is quite sensitive (good), but not as sensitive as PCR.

FeVL - developement of ANTIGEN (p27)

FIV - development of AB

52
Q

(Q11 b) Serological methods:
Immunodiffusion test
Procedure:

A
  • Agarose gel in petri plate, cutting 7 well like “rose”
  • Antigen in the middle
  • Positive control serum in one on the wells around
  • Four tested samples of serum in other wells
  • Application into well, incubation for 24-48 hours in room temperature.
  • Result in percipitation line= positive, because antigen reacted with antibody.
53
Q

(Q11 a) Serological methods: ELISA

Procedure:

A
  • Use microtitartion plate (96 wells)
  • Use spesific antigen (usually commersial) to cover each well and wait maybe 1 day so it’s stuck to the walls
  • Second day we add tested serum samples
54
Q

(Q11 c) Immunoblot

Procedure

A

Detection of virus,

  • Usually have some main protein from the virus wich we put on to the gel –> POLYACRYLE AMIDE GEL, and spread the virus by vertical electrophoresis. So proteins are separated.
  • Then proteins are blotted into a nitrocellulose membrane at 110 V for 1 hour - 1,5 hour.
  • After this the nitocellulose membrane stick on the gel
55
Q

(Q10) Tissue culture, cythopathic effect

Tissue culturing, proceedure

A

It’s 4 places we normally replicate virus in egg.

  • Use egg from breeder, that’s 9-11 days
  • Use ovoskop on egg to find and mark air sack and embryonic eyes.
  • Desinfect the shell with jodidum. Do everyting with mask and gloves.
  • Now egg is sterile and ready for inoculation of virus
  • Puncture air bubble by sterile needle
  • Put 0,2 ml of viral suspension
  • Close holes on egg with paste.
  • Incubate in thermostat for 3 days in 37 degrees,
  • After this, store egg in refrigirator for 12 hours, so embryo dies.
  • Then open egg (cut off the shell) and harvest virus from alantoic space (10-20 ml) with pipette.
  • After that: inoculate virus on blood agar and put into thermostat for 24 hours to check if any bacterial contamination. just to PROVE.

Influenza is very common to culture in egg, and it is done in allantoic space.

56
Q

(Q11 f) Hemagglutionation test (not serological test)
About it
Proceedure

A

Used for Orthomyxoviridae, Paramyxoviridae and Parvoviridae that contains HAEMAGGLUTININS (glycoproteins), which agglutinate erytrhocytes.

Avian influensa agglutinate hens RBC.

Procedure: Use plastic microtitration plate with 96 wells.
- Use sample from alantoic fluid (virus) and delute it times 2 (1/2, 1/4, 1/8, 1/16, 1/32.. osv), and prepare maybe 12 dilutions.
- The put the dilutions (12) on each own plate (12 wells alltogether).
- Add same amount of liquid of RBC.
- Incubate 2-4 hours in 4 degrees.
- POSITIVE RESLUT = HEMOLYSIS –> hemaglutination. HEMAGLUTINATION TITER= last well (virus dilution) where hemolysis happens.
NEGATIVE RESULT = one red dot in the middle = sediment of RBC, so virus didn’t ruin the bloodcells.

57
Q

(Q3) Fluorescent test

Procedure: (imp!)

A

Takes maybe 2 hours.

Inderect: Take a glass slide and add cell culture or tissue with KNOWN antigen.

  • Then add serum with antibodies e.g from commersial so you don’t know what it is but you sure know it will react.
  • Incubate in wet chamber in 37 degrees in 30-40 min.
  • Wash with PBS carefully. To remove any unbinded material.
  • Then add secondary antibody. This should be antibody against antibody (e.g. cat agianst cat). Must be same antibody origin!! Labeled with FITC.
  • Incubate again same way, wash with PBS, look in microscope, must be dry!

Slides can be kept refridigrated for like a month.

MICROBIOLOGY EXAM (2 decks)

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