Practical protocol 1 and 2

Differentation of bacteria

1. Preparation of Gram-stained smear

Prepare slide and apply Gram-staining according to teachers instructions. Note the result:

What characteristic features You can observe?

2. Ready smears. Observe and fill the gaps below.

a/ Congo-red staining

What You can observe?.....................................

b/ Ziehl-Neelsen staining

Application of staining………………………..

c/ Methylene blue staining (Loeffler)

Application of staning……………………….

3. Examination of hemolysis on sheep-blood agar

Describe the type of hemolysis and give an example of bacterial species name

Alpha………………………………….species…………………………………..

Beta…………………………………...species…………………………………..

Gamma………………………………. species…………………………………..

4. Catalase test

Note the result (presence/absence) for:

Staphylococci……………………………………..

Streptococci……………………………………...

Enterococci………………………………………

5. Examination of bacterial growth on differential and selective media:

6. Characterize culture media

7. Examination of growth requirements

Match the following bacteria: strict aerobic, facultative anaerobic, strict anaerobic with relevant arrows

8. Describe the application of following device

Candle jar- what type of bacteria may be cultured………………………………………………………

Anaerobic Gas-Pak jar – what type of bacteria may be cultured………………………………………..

9. Demonstration:

Determination of bacterial species using biochemical tests (API tests (visual reading), VITEK 2 Compact identification

cards)

Practical protocol 3

Differentation of fungi

1. Preparation of Gram –stained direct smear from oral cavity. Search for fungal cells. Note the result………………..

2. Preparation/observation of Gram –stained smears of different species of Candida cultured on Sabouraud medium.

Answer: are there any significant differences between fungal cells of each species?.......................................... ..........

3. Examination of direct Gram –stained smears from vaginal candidiasis (as an example).

Answer: when and how we can determine the yeast infection basing on the microscopic observation of smear?

………………………………………………………………………………………………………………

4. Yeasts – fill the table below

5. Examination of Candida albicans and non - Candida species in germ- tube test.

Note the result for:

Candida albicans - ………………………………………

Non-albicans Candida species-………………………….

Application of test………………………………………………………………………………………………

Positive result of germ-tube test

6. Molds (on Aspergillus sp. example) – fill the table below

7. Observation of hypha/mycelium of molds and dermatophytes in lactophenol cotton blue slide mounts.

Answer: What is the diagnostic role of slide mounts in identification of filamentous fungi?..........................................

……………………………………………………………………………..

8. Dermatophytes

Observe the growth characteristics of 2 different species of dermatophytes. Note Your observations.

1.Species…………………………………………….

…………………………………………………………………………………………………..

2. Species……………………………………………

…………………………………………………………………………………………………..

9. Dermatophytes – fill the table below

Practical protocol 4

Viruses – isolation and detection

1. Slide haemagglutination assay – self performance

Draw/describe the result

Application of the test……………………………………………………….

2. Tube haemagglutination test

3. Rabies virus in brain tissue – direct fluorescent smear

Draw/describe the result

4. Plaque assay – the determination of the concentration of infectious phage particles

Draw/describe the result ……………………………………………………………………………………….

Application of the test…………………………………………………………………….

5. Answer:

What is cytophatic effect?......................................................................

Which tissue and how presents the presence of the following viruses?

CMV-…………………………………………………………

Adenoviridae - ………………………………………………..

Rabies virus - …………………………………………………

Other…………………………………………………………..

Practical protocol 5

Differentation of Gram – positive bacteria

1. Differentation of staphylococci

2. Differentation of streptococci

3. Differentation of enterococci

4. Corynebacterium species – observation of ready Gram- stained smear.

Note Your observation…………………………………………………

5. Mycobacterium species - observation of ready Ziehl – Neelsen – stained smears (acid-fast)

Note Your observation …………………………………………………

- observation of growth on Loewenstein Medium

Note Your observation…………………………………………………..

- application of Real Time PCR (Mycobacterium DNA detection).

6. Observation of Bacillus cereus on sheep blood agar

Note Your observation……………………………………………………

Practical protocol 6

Differentation of Gram – negative bacteria

1. Culture own specimen (at least 2 different) collected from chosen body site on appropriate solid media according to

Assistant’s instructions and hints provided below.

NOTE: consider checking Yourself for being a carrier of S. aureus in prenares.

2. Differentation of Moraxella sp. and Neisseria sp.

3. Note the results of observation of growth of Gram-negative rods on solid media:

Answer:

a/ Which Gram-negative bacteria we call “non-fermenters” and why?

............................................................................................................................. ......................................................

b/ How are the bacterial features determined with using McConkey agar accelerate the diagnostic? How it helps a (doctor) patient

infected with those bacteria?

………………………………………………………………………………………………………………….......

4. Demonstration of biochemical kits for full identification of Gram – negative bacteria (API ID 32 E, VITEK GN).

Application to determine………………………………………………………………………………….

5. Demonstration of kits for serological typing of Salmonella and Shigella.

Application of serology……………………………………………………………………………………

Answer: Why serology is reccomended method to determine the presence of Salmonella and Shigella species?

…………………………………………………………………………………………………………….

Practical protocol 7

Diagnostic of anaerobic infections

1. Examination of bacteria in students’ cultures, inoculated at practical class no 6.

Observe and note in the table the results of cultures performed from different body-sites. Consider the representatives of

humans microbiota basing on the features of used solid media.

Answer:

How and when should be the further diagnostic performed (in case of normal flora, carrier state, infection, contamination)?

Which of the obtained results reflect the normal human biota in selected body site?

2. Self - prepared Gram-stained smears form gingival pocket or dental plaque (according to Assistants instructions).

Note the results of microscopic observation.

3. Observation of Clostridium perfringens Gram stained smear (swab from wound of patient with gas-gangrene).

Note the result

4. Observation of Propionibacterium acnes and Clostridium perfringens on Schaedler medium.

Note characteristic features of bacteria:

……………………………………………………………………………

……………………………………………………………………………

5. Diagnostic of infection caused by Clostridium difficile. Antigen detection is stool sample.

ANSWER

What is detected using demonstrated test?

Basing on the picture answer - if the result is positive/negative

Is culturing of Clostridium difficile from stool sample an essential element of diagnostic?

Practical protocol 8

Diagnostic of fungal infections

1. Analyze the reports of serological diagnostic of fungal systemic infections – mannan and galactomannan detection.

Note the essential elements provided on results/s:

…………………………………………….

Answer:

What is the method recommended to determine the presence of fungal antigens?...................

What specimen is collected?.....................................................

2. Detection of DNA of Aspergillus sp. and Pneumocystis jiroveci from clinical samples – demonstration of the results.

Note the essential elements provided on printed results/s:

…………………………………………………………………………..

Answer:

What is the method used in this type of analysis?............................

What specimen is/may be collected?..................................................

What type of infections are caused by detected microorganisms?...........................................

3. Determination of susceptibility of Candida species to antifungal agents. Read and interpret the results of susceptibility

testing (Candifast results).

Note the results:

Species name:…………………………

Susceptible to:…………………………

Resistant to:…………………………….

Answer

1. How do we determine the susceptibility of molds and dermatophytes to antifungal agents?......................................

Practical protocol 9

Diagnostic of viral infections

According to Assistants instructions

1. Examination of own fingers before handwashing, after handwashing using soap, after disinfection – self-

preparation.

2. Examination of microbial air contamination by means of spontaneous sedimentation – self preparation.

3. Examination of contamination of different surfaces by means of Count-Tact plates and swabs – self

preparation.

4. Interpret the results of serological markers of HBV infection according to disscussion with the Assistant

and provided table. (source: https://www.cdc.gov/hepatitis/hbv.)

5. Analyze the reports of serological diagnostic of viral infections .

Detection of DNA CMV and BK virus in patient’s serum by real-time PCR .

Note the essential elements of printed result/s:

………………………………………………………………..

6. The quantitative determination of antibodies against Epstein-Barr virus (IgM and IgG) by Luminex Map

Technology – analysis of lab reports.

7. Fill the gaps of the table

Virus Test

name

What is

detected

Clinical

application

Name of

disease/

syndrome

Treatment(causal/symptom

atic)

HIV

CMV

Influenza

virus

EBV

RSV

Rotavirus

HPV

BK