Nursing Management : Methods for Laboratory Diagnosis of Bacteria

Methods for Laboratory Diagnosis of Bacteria

Specimen collection and Examination of Bacteria:

- Specimens are collected with a blade or by swabbing the involved areas of the skin using a sterile dry cotton wool.

- If the tissue is deeply ulcerated, or if pustules and blisters are present, aspirate a specimen using a sterile needle & syringe.

- The purulent discharges or exudates are spread as thinly as possible on a glass slide for Gram staining.

- After collecting the specimen with the swab, insert the swab in to a sterile tube for culturing.

- For actinomycetes, pus is collected from closed lesions by aspiration with a sterile needle and syringe. Material is collected from draining sinuses by holding a sterile test tube at the edge of the lesion & allowing the pus & granules to run in to tube. Granules are aggregates of inflammatory cells, debris, proteinatious material & delicate branching filaments. Pus & other exudates are examined for the presence of granules.

a. Examine the specimen microscopically

Gram smear

1. Make an evenly spread smear of the specimen on a slide
2. Allow the smear to air-dry in safe place
3. Stain the smear with the gram technique
4. Examine the smear for pus cells and bacteria. Mostly skin infection causing bacteria can be differentiated by their Gram reaction due to difference in their cell wall structure.

- Gram positive Cocci that could be S. aureus
- Gram positive streptococci pyogens or pneumonia
- Gram negative rods that could be P.aeruginosa, proteus species, E.coli, or other Coliforms
- Gram variable rods lying in chains that could be B.anthracis.
Gram-negative coccobacilli that could be Y. pestis.

b. Examine the specimen using culture

Blood agar and MacConkey agar cultures are used for isolation of bacteria, which cause common skin diseases.

- Look for colonies like

o Staphylococcus aureus
o Streptococcus pyogenes
o Pseudomonas aeruginosa
o Enterococci
o Proteus species
o Escherichia coli

- Modified Tinsdale medium culture could be used if cutaneous diphtheria is suspected

- Use room temperature for Blood agar and MacConkey agar if Yersina pestis is suspected.

Culture the specimen

- Flame and sterilize wire loops before & after use

- Flame the necks of specimen bottles, culture bottles, & tubes after removing & before replacing caps.

- Inoculate the culture media

- Make slide preparations from specimens after inoculating the culture media

- The inoculated media should be incubated as soon as possible. Incubate the blood agar & MacConkey agar aerobically at 350 – 370C.

- After 2 days and onwards of incubation examine both cultures for the common bacteria of skin infection.

- Report the identified bacterial from the cultureGram-negative coccobacilli that could be Y. pestis.

b. Examine the specimen using culture

Blood agar and MacConkey agar cultures are used for isolation of bacteria, which cause common skin diseases.

- Look for colonies like

o Staphylococcus aureus
o Streptococcus pyogenes
o Pseudomonas aeruginosa
o Enterococci
o Proteus species
o Escherichia coli

- Modified Tinsdale medium culture could be used if cutaneous diphtheria is suspected

- Use room temperature for Blood agar and MacConkey agar if Yersina pestis is suspected.

Culture the specimen

- Flame and sterilize wire loops before & after use

- Flame the necks of specimen bottles, culture bottles, & tubes after removing & before replacing caps.

- Inoculate the culture media

- Make slide preparations from specimens after inoculating the culture media

- The inoculated media should be incubated as soon as possible. Incubate the blood agar & MacConkey agar aerobically at 350 – 370C.

- After 2 days and onwards of incubation examine both cultures for the common bacteria of skin infection.

- Report the identified bacterial from the culture

To dispatch skin specimens to a microbiology laboratory:

1. Collect the specimen using a sterile cotton wool swab. Insert in a contained of Amies transport medium.

2. Make smear of the material on a clean slide & allow to air-dry in a safe place

3. Label the specimens using a lead pencil. Mark the specimen HIGH RISK if from a patient with suspected anthrax or Bubonic plague (Yersina pestis).

4. Send the specimens with a request form to reach the laboratory within about 6 hours.

Gram staining technique

- Required reagents;

o Crystal violet stain
o Lugol’s iodine
o Acetone – alcohol decolorizer
o Neutral red – 1g/l (0.1% w/v) or safranin

Method

1. Fix the dried smear.

Note: when the smear is for the detection of Gonococci or Meningococci, it should be fixed with methanol for 2 minutes (this avoids damaging pus cells)

2. Cover the fixed smear with crystal violet stain for 30-60 seconds

3. Rapidly wash off the stain with clean water

Note: - when the tap water is not clean, use filtered water or clean boiled rainwater

4. Remove excess water, and cover the smear with Lugol’s iodine for 30-60 seconds

5. Wash of the iodine with clean water

6. Decolorize rapidly (few seconds) with acetone – alcohol wash immediately with clean water
Caution –Acetone alcohols are highly flammable therefore use it well away form an open flame

7. Cover the smear with neutral red (safranin) stain for 2 minutes

8. Wash off the stain with clean water

9. Wipe the backs of slide clean, and place it in a draining rack for the smear to air-dry.

10. Examine the smear microscopically, first with the 40-x objective to check the staining and to see the distribution of material and then with the oil immersionobjective to report the bacteria and cells.

Results:

- Gram positive bacteria ……………………………… Dark purple
- Yeast cell ………………………………………………Dark purple
- Gram negative bacteria …………………………….. Pale to dark red
- Nuclei of pus cells …………………………………… Red
- Epithelial cells ……………………………………….. Pale red

Reporting gram smears

The report should include the following information;

- Numbers of bacteria present, whether many, moderate, few or scanty,
- Gram reaction of bacteria, whether gram positive or gram negative
- Morphology of bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli. Also, whether the organisms are intracellular or extra cellular.
- Presence and number of pus cells
- Presence of yeast cells and epithelial cells

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Nursing Management : Methods for Laboratory Diagnosis of Bacteria 2018-12-26T00:39:00-08:00 Rating: 4.5 Diposkan Oleh: David Maharoni