Bacterial diagnostics

Question 1

1. PREPARATION AND FIXATION OF BACTERIAL SMEARS

6 steps.

Answer 1

** The used slide has to be clean and without any fat on its surface - To achieve this, flame the slide 5 times above the Bunsen flame.
** Microorganisms are killed ( No motility, metabolism or reproduction can be observed)
1. Liquid sample: With the loop smear a small droplet of the sample (gently agitated broth culture) on the slide and spread it evenly within a circle the size of a nickel. 2. Solid sample: Bring a small drop of top water solution to the surface of the slide. Transfer and suspend a small amount of a surface growth from sample (slant agar, Petri dishes) with loop.
3. Let the sample dry at room temperature
4. Identify the slide on the back side with a marker
5. Fix the sample with flaming the slide ( sample side up ) above the Bunsen flame 3 times (Other fixation technique: e.g. methanol)
6., Staining (supravital)
 Loops always should be burned before and after use

**Simple stain- just one stain is used e.g methylene blue.
**Complex stain: we use more than one dye, so Differentiation is possible, e.g:
. Gram-, Neisser-, Ziehl-Neelsen (acid-fast) staining

Question 2

2. Native preparations and their application. Vital staining methods and their applications.
3 types:
first type is 3 steps
second type is 5 steps
last stain is 4 steps

Answer 2

NATIVE PREPARATIONS (unstained preparation examined by bright field, dark field, or phase-contrast microscopy)
Which allows:
• examination of living microbes
• presence or absence of microbes in specimens directly
• morphology (shape)
• motility
• metabolism of microbes

Types:
A . Wet-mount preparation

1. Place a drop of bacterial or protozoa suspension with pipette or loop in the centre of the slide Or Disperse a small quantity of the specimen to be examined into a drop of saline on a microscope slide
2. Cover with the coverslip
3. Examine immediately the slide under low power /40 X objective, with lowering the condenses/

B. Hanging-drops preparation

1. Place a drop of bacterial or protozoa suspension with pipette or loop in the centre of the slide
2. Spread with Vaseline the rim of the hollow slide’s cavity.
3. Carefully invert this slide over the coverslip that the inoculums’ drop should be just under the centre of the well.
4. Fast reverse
5. Examine the slide under low power ⇒ The motile microbes are those which dart through the microscope field ⇒ This is in contrast to the random Brownian motion which all microbes exhibit

C. Vital staining

1. Make a wet-mount preparation
2. One drop of diluted methylene blue stain or Lugol’s iodine to the edge of the coverslip
3. Wait 10 minutes
4. Examine the slide under low power ⇒ The dyes non-specifically stain the cellular material, increasing the contrast with the background (e.g. bacterial cells-methylene blue), and permit examination of the detailed structures (e.g. Lugol – parasitology)

Question 3

gram stain

Answer 3

1. , Fixation by heat
2. , Cristal violet + sodium-oxalate solution 2’
3. , Iodine + potassium-iodide solution 1’
4. , Differentiation by 96 % ethanol until colourless drops!
5. , Fuchsin/safranin 1’
6. , Drying with filter paper
7. , Microscopical examination with immersion

Gram-positive: deep purple Gram-negative: pink Unstainable by Gram
Cystal violet= purple color
Safranin= red.
The mechanism of the Gram stain appears to be generally related to the thickness of the cell wall, pore size, and permeability of the intact cell envelope.

Question 4

4. neisser stain

Answer 4

1.Stain fixed smear of cultured Corynebacteria with a freshly prepared 2:1 mixture of Neisser I and Neisser II dyes for 10 minutes.
2. Rinse the slide with water.
3. Strain the smear with crysoidine dye for 2 minutes.
4. Removing the excess of dye
5., Drying with filter paper
6, Microscopical examination with immersion

Rods with club ends the body of which strains yellow and end stain, metachromatically purple (volutin granula = poli-phosphate)

Cells arranged in a manner like Chinese characters

Question 5

5. Acid-fast staining (steps and mode of action)

6 steps…

Answer 5

Used to stain mycobacteria and other acid-fast organisms.

Mycobacteria have a Special cell wall; rich in lipids:
So they do not stain by Gram.

Steps of Ziehl-Neelsen (hot acid-fast) stain:
1. fix smear.
2. cover with carbol-fuchsin (with the help of filter paper)
3. steam 3 times (uptake of carbol-fuchsin requires heating specimen)
4. Differentiation: acidic alcohol
3% HCl, 96% ethanol
“acid fast”.
5. Wash with water!
6. counter stain 2 min with Loffler’s methylene blue

Organisms appear RED (RODS) – against light-blue background

Question 6

6. Detection of bacterial capsule, spore and flagella

Answer 6

CAPSULE: India Ink—negative staining!, antibodies (Kantigen); Neufeld quellung-reaction

1. , Degrease and cool the slide
2. , 1 drop of India Ink onto the end of slide
3. , Mix the sample
4. , Disperse the drop by another slide
5. , Drying
6. , Fixation
7. , Possible staining (fuchsin/methylene blue)
8. , Microscopical examination with immersion.

SPORE:

1. Ziehl-Neelsen.
2. Gram (lack of staining).
3. Special: malachite-green or carbolfuchsin.

 FLAGELLUM: silver impregnation, antibodies (H-antigen)

Detecting Bacterial Motility /flagella

since motility is a primary criterion for the diagnosis and identification of bacteria, several techniques have been developed to demonstrate bacterial motility, directly or indirectly:
1. Flagella stain, cannot be seen in light microscope. However, their presence and arrangement can be demonstrated by treating the cells with unstable colloidal suspension of tannic acid salts
… 3. silver impregnation,
2. antibodies (H-antigen)

Question 7

7. Significance and information content of microscope examinations..

Answer 7

In general, microscopy is used in microbiology for two basic purposes: the initial detection of microbes and the preliminary or definitive identification of microbes.

 Quality, quantity and proportion of microorganism in the sample
 Presence or lack of normal flora
 Morphology: characteristics of cells and their arrangement
 Size
 Morphology
 Intra/extracellular localisation
 Cell wall (Gram)
 Spore (Ziehl-Neelsen), capsule (India Ink, Neufeld), flagellum (silver impregnation): presence or lack
 White blood cells (+ other types of cells): presence or lack.

MICROSCOPIC EXAMINATIONS
1. Light (bright field) microscope -
resolving power: 250nm.
Maximal magnification: 1500 X oil immersion ⇒ Bacteria, fungi, protozoa

2. Electron microscope - transmission electron microscope - scanning electron microscope
   Resolving power: 0,1 nm - ⇒ Viruses
3. Dark field Illumination - special condenser –→ „dark field „ contrasts against the highlighted edge of the specimens - for investigation of Spirochetes ( treponema, borrelia, leptospira), which are less than 0,2 um in diameter, and therefore cannot be observed with direct light

Question 8

8. Control of sterilization and sterility

Answer 8

wtf..