1. Practical: Revision
Dr. Salma Elamin

2. Safety in the Laboratory
A safety updated manual should be available in the laboratory, to regulate proper handling of infectious and hazardous materials, as well as proper using and handling of equipments.
Emergency procedures should be known to all laboratory personnel.
Lab-coats should be worn in the laboratory at all times.

3. Safety in the Laboratory
Learn the location of first aid boxes, and fire fighting equipments.
To avoid the risk of transmitting infectious pathogens when working with blood specimens, precautions must be taken.
Proper hand wash procedure to be followed before and after any procedure or wearing gloves:
1. Wet hands and wrists with water
2. Apply soap to the palms from a dispenser.

4. Safety in the Laboratory
3. Rub both hands, wrists, fingernails, and between fingers, well with soap, for a minimum of 5 seconds. 4. Rinse well with water and dry. 5. Sinks with foot – operated controls are ideal, when not available try to use a paper towel to turn off water so that clean hands would not be contaminated again.

5. Safety in the laboratory
When working with a potentially infectious biological material, procedures to follow:
1. Never mouth- pipette.
2. Handle infectious fluids carefully to avoid spilling and avoid occurring of aerosols.
3. Avoid using needles and syringes unless necessary. Dispose of sharps in the designated container.

6. Safety in the laboratory
4 Use protective gloves and laboratory.
5. wash hands frequently, after taking of the gloves, after laboratory activities, and after coming in contact with an infectious material.
6. Decontaminate surfaces before and after use. Wipe any spills immediately.
7. Never eat , drink, or smoke in the laboraty.

7. Laboratory Waste Disposal
Infectious waste: e.g. Blood, blood products, sharps, microbiological waste, should be packed in for disposal in color coded containers labeled with the universal symbol for biohazards .
Containers for waste must be easily accessible and not to be over filled.

8. LABORATORY EQUIPMENT & Media
Incubators
Temperature is set as required e.g.: 37˚C
CO² Incubator
Are device for controlling the temperature, humidity, and other conditions in which a microbiological culture is being grown. Incubators can vary in size from tabletop to units the size of small rooms.
Temperature is set as required (optimum temperature) e.g.: 37˚C
Incubators with CO₂ or O₂ gas are used according to growth requirements of the organism to be cultured.

9. Biological safety cabinet
Cabinet Purpose
In varying degrees, a laminar flow biological safety cabinet is designed to provide three basic types of protection.
Personnel protection from harmful agents inside the cabinet.
Product protection to avoid contamination of the work, experiment or process.
Environmental protection from contaminants contained within the cabinet.
How Biological Safety Cabinets Are Classified”
Classification Biosafety Level ApplicationClass I1,2,3low to moderate risk biological agentsClass II1,2,3low to moderate risk biological agentsClass III4high risk biological agents

10. Hot Air Oven: Sterilization by Dry Heat

11. General Biohazard Sign - equipment
Biohazard sticker/label, that is red or orange in colour with a biohazard symbol and lettering in black as illustrated.
It identifies equipment/instrument containing biological materials: refrigerators, sharps containers, storage areas, freezers, centrifuges and wastes containers. DO NOT TOUCH.

12. Safety Goggles
Disposable Mask:

13. Inoculating Loops and Needles
Petri Plates or Petri Dishes:

14. Compound light microscope:

15. Forms of Culture Media:

16. 1-Simple Stains Examples of simple stains are:
a- Loffler’s methylene blue: the most valuable reagent available for staining bacteria.
It is excellent for the genus Corynebacterium, it can demonstrate beading, barring, and granules.
With sporing organisms stained with methylene blue, spores appear as unstained bodies within the cell.

17. 1-Simple Stains To stain a slide with methylene blue:
1. Apply stain for 1 minute.
2. Rinse with water.
3. Drain or blot dry.
Methylene blue staining demonstrating the typical morphology of Corynebacterium diphtheriae

18. Gram staining
The color of the bacteria at the end of the gram-staining procedure (either purple or pink) depends on the chemical composition of the cell wall.
Gram-positive bacteria retain the purple color of the crystal violet.
In Gram-negative cells, the crystal violet is removed during the decolorization step and the cells are subsequently stained pink by the safranin.
Some strains of bacteria are neither consistently purple nor pink, following this procedure. They are referred to as Gram- variable bacteria.

19. Gram Stain Components of Gram Stain:-
Crystal violet (The primary stain).
Iodine (fixative or mordant).
Acetone or Alcohol (the decolorizer).
Safranin ( the counter stain).

20. Steps of Gram Stain
1.The heat-fixed smear is covered with a solution of the basic dye: crystal violet (a purple dye) for 1 minute.
2. Rinse gently with water and cover the smear with Gram’s iodine solution (fixative or mordant) for 1 minute.

21. Steps of Gram Stain
3. Wash off the iodine with water and decolorize with acetone (Decolorizer) for 1-5 seconds, if the decolorizer is left longer on the slide it can wash of the primary stain off.
4. The slide is counterstained with safranin (a bright red dye) for I minute, rinse, dry, and examine using the oil immersion objective.

22. Gram stain of Lactobacillus species
Illustrating gram positive bacilli, single and in chains
Gram stain of Escherichia coli
Illustrating short gram negative bacilli

23. Bacterial Spores (Endospores)
Endospores are formed by a few groups of Bacteria as intracellular structures, but ultimately they are released as free endospores.
Biologically: endospores show no signs of life.
They are highly resistant to environmental stresses such as high temperature (some endospores can be boiled for hours and retain their viability), irradiation, strong acids, disinfectants, etc.
They are probably the most durable cell produced in nature.

24. How does the Stain Works:
It is difficult to get a dye into an endospore because of its low penetrability and high degree of resistance due to multiple coats surrounding the spore.
After applying the primary stain, the slide is heated over a steam bath to soften the hard outer layer of the cell, and allow the Malchite green to bind to within the spore.

25. How does the Stain Works:
This stain cannot be decolorized from the spore once they are removed from the heat ; however water can remove the Malachite Green from the vegitative cells making them colorless again.
When we apply Safranin as the counter stain, the vegetative cells take up this stain and appears red/pink.

26. Spore Stain: A Special Stain Schaeffer- Fulton Procedure
Spore Stain Method: - Flood the slide with 5% aqueous malachite green and steam for 1 min. - Wash under running water. - Counterstain with 0.5% aqueous safranin for 15 seconds. - Rinse with water and drain or blot dry. Bacterial bodies stain red, spores green. 26

27. Spore Stain Showing spores of Bacillus subtilis stained green by Malachite Green

28. Capsule Staining
Capsules are not usually stainable by basic stains, because of their chemical characteristics.
They are best observed by a procedure called negative staining.
Two forms of negative staining are used for capsule detection: a. Uses India Ink and wet preparation.
b. Uses India Ink followed by smearing and drying on the slide.

29. Negative Differential Stain Method (India Ink)
Procedure:
India ink wet-film staining method for capsules:
- This is a negative stain: colouring the background so that the cells are shown as clear objects. Performed as follows:
1. Place a large loopful of undiluted India ink on a slide.
2. Mix this with a small portion of the bacterial colony or a small loopful of the deposit from a centrifuged liquid culture.
3. Place a coverslip on top and press down under a pad of blotting paper.
The capsule appears as a clear light zone between the refractile cell outline and the dark background.

30. How Does India Ink Stain Works& Why Is It Called: Negative Stain
India ink is composed of fine carbon particles that are suspended in water and that form a true colloid(do not settle out of suspension).
These particles are too large to penetrate the gel-like matrix of the capsule.
When a drop of India Ink is mixed with a drop of broth culture (when seen by light microscope), the capsule and cells are seen as transparent zones of different density, surrounded by a dark (India Ink) background.
So the capsule and cell are observed indirectly because they exclude the carbon particles: Negative Stain.

31. Bacterial capsules outlined by India ink viewed by light microscopy.
This is a true capsule, a discrete layer of polysaccharide surrounding the cells. Sometimes bacterial cells are embedded more randomly in a polysaccharide matrix called a slime layer or biofilm.

32. MYCOBACTERIUM
The most obvious characteristic of the mycobacteria is the large amount of lipid presents in their cell walls−approximately 40% of the total cell dry weight−causing them to grow as extremely rough, hydrophobic colonies.

33. MYCOBACTERIUM
Mycobacteria are also difficult to stain, but once stained, they resist discoloration.
Organisms with the ability to retain a stain in spite of washing with acid alcohol are referred to as acid-fast.
Only the members of the genus Mycobacterium and a few species of Nocardia possess this property and this characteristic helps in detecting mycobacteria in body fluids such as sputum.

34. Differential stains
Differential staining procedures have two components: a differential stain, followed by a simple stain.
The dyes used in each are different colors, so that a positive cell will be one color and a negative cell another.
Acid-fast stain is a differential stain

35. Ziel- Neelsen stain (Acid-fast)
To get the dyes into these cells, it is necessary to employ methods such as heating the organisms in the stain or including detergents in the stain.
However, once these cells are stained by carbolfuchsin (a mixture of phenol and the dye fuchsin), their cell-wall structure allows them to retain the stain even when washed with 95% alcohol containing 3% HCl−hence, the term acid-fast. All other bacteria are decolorized by this procedure.

36. Ziel- Neelsen stain Method
Ziehl- Neelsen’s method (Acid- fast stain):
1. Flood the slide with strong carbol fuschin and heat until steam rises (but do not boil).
2. After 3-4 min apply more heat until steam rises again; do not let the stains dry on the slide.
3.About 5-7min after first application of heat, wash the slide thouroughly under running water.
4. Decolorize with acid-alcohol until all traces of red have disappeared from the film.
5. Wash well in water when decolorization is complete.
6. Counterstain with Loeffler’s methylene blue or 0.5% malachite green for 1 min.
7. Wash and stand on end to drain : DO NOT BLOT.

37. Acid-fast stain ( carbolfuschin based) sputum smear Showing the presence of Mycobacterium species stained by Ziel-Neelsen method