YARMOUK UNIVERSITY (2020)

By: Yousef Alzoubi

Micro lab (1-4)
Laboratory safety : Biological Safety Cabinet (BSC) :
 Clinical laboratory staff (& potentially the public) exposes to  BSC is a device that encloses a workspace in such a way as to
a variety of biological, chemical and physical hazards protect workers from aerosol exposure to infectious disease
 Everyone in microbiology lab must follow specific safety rules agents
and procedures  Air that contains the infectious material is sterilized, either
1. Wear protective clothing PPE (PERSONAL PROTECTIVE by heat, UV light, or, most commonly, by passage through a
EQUIPMENT ) which include (glasses or goggles , lab coat HEPA filter (High Efficiency Particulate Air)
or long sleeves , long pants , gloves , closed-toe shoes)
2. Avoid touching objects (e.g., pencils, cell phones, door  Biological Safety Level (BSL) :
handles) while wearing gloves These cabinets are designated by
3. Pencils, labels, or any other materials should never be classes (1-4) according to the
placed in your mouth degree of biologic safety they
4. Caution must be taken when using gas burners afford .
5. Long hair and hijabs must be tied back
6. Do not eat food or drink water in the lab (do not use lab
glassware as food or water containers) BSL – 4 BSL- 3 bsl – 2
7. Protect your hands - Wash hands after every lab
8. Do not take any culture media out of the lab for any
reason (All cultures should be handled as potentially
pathogenic)
9. DO NOT ENGAGE IN PRACTICAL JOKES IN THE LAB!
10. Wipe the bench tops with disinfectant (70 % Alcohol)
before and after work
11. Report all accidents (no matter how minor) to your
supervisor
12. Dispose of waste products according to instructions
 Biological Safety Level (BSL-1) : Microscope: is an instrument used to magnify (visually enlarge)
 Microbes Not known to cause disease in healthy adult objects which are too small to be seen by naked eye ,so that
humans, Bacillus subtilis their characteristics can be observable
 Safety equipment: Minimal requirements Types of microscopes :

 Biological Safety Level (BSL-2) : 1- Simple microscope (composed of a single lens)

 Associated with agents that cause mild to moderate 2- Compound microscope (composed of two or more
infections e.g. Salmonella, E.coli, Influenza viruses lenses)
 Safety equipment: Biological Safety Cabinet and  Brightfield microscope
personal protective equipment as needed  Darkfield microscope
 Phase-contrast microscope
 Biological Safety Level (BSL-3) :  Fluorescence microscope
 Agents associated with serious human diseases and 3- Electron microscope (EM)
with potential for aerosol transmission (inhalation
BRIGHT-FIELD MICROSCOPE :
route) – e.g. M. tuberculosis , SARS , Anthrax
 Safety equipment : Biological Safety Cabinet and  Illumination source : Visible light (tungsten lamp)
personal protective equipment required  Nearest to eye, there is ocular lens (eyepiece) with a
magnification of x10
 Biological Safety Level (BSL-4) :  nearest to specimen, there is 4 objective lenses
 Dangerous agents of life threatening nature – e.g. (Scanning 4x , Low 10x, High 40x and oil immersion 100x)
Ebola virus, HIV  total magnification = objective x ocular lens magnifications
 Safety equipment
- Biological Safety Cabinet
- Full-body air-supplied,
- positive pressure personnel suit
 The most common type microscope used in the clinical field
 Magnification of 1000× allows for optimal visualization &
characterization of fungi, parasites, & bacteria but is
insufficient to visualize internal structures and viruses
 Limitation: absence of contrast between objects &
background of specimen
 Contrast is achieved by staining techniques that highlight
organisms and allow them to be differentiated from the
background
 Used for non-viable (non-living ) , stained preparations
 Opaque image againest brighter background

Parts of the microscope :

 Nose Piece : holds objective lenses & can be turned to
increase the magnification . When the lens is in the right
position you will hear (click) sound
 Stage/Stage Clips : A fixed platform for the placement of the
specimen, with an opening in the center allows light passage
from the illumination source to the lens system . Two stage
clips hold the slide/specimen on the stage
 Diaphragm : Controls the amount of light entering the lens
system
 Coarse Adjustment Knob : Moves the stage up & down
for bringing the image into focus , Move very slowly until
you see an image
 Fine Adjustment Knob : to sharpen or clear the image
after using the coarse adjustment knob
 HINT: when there is no image use coarse adjustment
when there is unclear image use fine adjustment
 Why do we add oil when we use the oil immersion lens?
a drop of immersion oil is placed between specimen and
the lens to reduce scattering of light
 RI (refractive index) of air < glass → as light rays pass from
glass into air → they refract → so they do NOT pass to
objective lenses → this will cause loss of light
RI of oil = glass ; preventing light rays from dispersing &
changing wavelength after passing through the specimen
General guidelines for using the microscope :
1. Turn on your microscope 6. Rotate the coarse adjustment knob until you see the
2. Clean the lenses by lens paper image

3. Place the slide over the center of the stage 7. Rotate the fine adjustment knob until the image is clear

4. Adjust the light 8. Switch to the next powerful objective lens and make final
focus adjustments
5. Rotate the revolving nosepiece to the lowest power
objective lens 9. Add a drop of oil when using the oil immersion lens
10. Examine your specimen!
 Storage and care :  Basic Stain :
 Always carry microscope with 2 hands  Creates Cationic (+ve ) chromogen & Works best in basic pH
 Clean oil immersion lens with xylene or absolute Alcohol Examples:
 Clean the lenses by lens paper 1. Carbol Fuchsin (red or pink color)
 Always store covered 2. Methylene Blue
 store on the lowest magnification 3. Malachite Green
 Remove the slide 4. Crystal Violet
 Turn off microscope 5. Safranin (red or pink color)
 Acidic Stain :
Staining of microorganisms :  Creates Anionic (-ve ) chromogen & Works best in acidic pH
 Examples:
 Stains (dye): are chemicals used to increase contrast 1) India ink (deep blue color) 2) Eosin (red color)
between organisms & the background to be microscopically 3) Nigrosin (deep violet) 4) Picric acid (yellow color)
easily visible
 STAIN is a COLORED CHARGED compound Smear preparation :
 Chemical formula:  A milky suspension of bacteria and distilled water
1- Benzene ring = colorless organic compound  The preparation of a smear is required for many lab
2- Chromophore = color procedures, e.g. staining
3- Chromogen = Charge  The purpose of making a smear is to kill and fix the bacteria
 Types of Stains (based on the charge of chromogen): onto the slide by heat
1- Basic stain (+ve) 1) Place one loop of solid bacterial growth or two loops
2- Acidic stain (-ve) of liquid bacterial growth in the center of a clean slide
 The bacteria’s cytoplasm has a negative charge
2) If working from a solid medium, add one drop of distilled
water to your specimen. (don’t add water with broth
samples)
3) Mix the specimen with the water and spread the
suspension over about half of the slide
4) Wait for the slide to dry (2-3 min)
5) Heat fixation, pass the slide quickly over bunsen burner
flame (3-4) times , this step will kill the bacteria and fix it to
the slide to avoid losing it during washing steps in staining
techniques
Staining methods :
A- Simple staining uses single (only one) stain
 Direct staining
 Indirect staining
B- Differential staining uses multiple (≥2) stains
 Gram’s stain
 Acid fast stain
 Cellular structures (capsule, endospores)
Simple – Direct staining : (colored bacteria)
 Uses Basic stains
 Basic stain (+ve charge) & Bacteria (-ve charge) so,
attraction between stains , stain gets inside bacterial cell
wall making them colored against a clear background
 Flood smear with basic stain for 30-60 seconds, then rinse
with water, blot dry, examine under oil immersion lens
Simple – Indirect staining : (colorless bacteria )
 Uses Acidic stains
 Acidic stain (-ve charge) & Bacteria (-ve charge) so,
repulsion , stain stays out bacterial cell making them
uncolored against a colored background
 Used commonly to show: - capsules - natural shape and
size
 This method DOES NOT need smear preparation
Differential – Gram’s staining :
 Most commonly used for microscopic examination &
identification of bacteria
 Used to divide bacteria into two groups:
Gram positive - appear purple
Gram negative - appear pink/red
 G+ve: Thick peptidoglycan layer while G -ve thin
peptidoglycan layer with outer membrane (
lipopolysaccharide layer)
Gram’s staining reagents :
1) Primary stain - Crystal Violet
2) Mordant - Gram’s Iodine
3) Decolorizer - Ethanol / Acetone
4) Counter stain - Safranin
Gram’s staining procedure :
1) Flood the fixed smear with crystal violet, allow to stand
for 1 min
Crystal violet (basic stain +ve) enters to both cells and
stains them purple

2) Flood the slide with Iodine, allow to stand for 1 min

Iodine enters to both cells creating (crystal violet – iodine
complex) in both cells / color remain purple for both

3) Drip the acetone-alcohol across the slide for 15 sec

(critical step)  Bacteria that retains the color of the primary stain is
Decolorizer has no effect on the peptidoglycan layer of G+ve
G+ve cell wall, remain purple  Bacteria that retains the color of the counter stain is
Decolorizer dissolves the Lipopolysaccharides layer of G- G-ve
ve cell wall, (CV-I complex) will be washed away from the
cell leaving it colorless
4) Flood the slide with safranin, allow to stand for 1 minute
Safranin enters both cells, making the colorless G-ve cell
pink. While it can’t mask the purple color of G+ve cell so it
stays purple
5) Blot dry & examine under Oil Immersion
 Differential – Acid Fast staining :
 AKA: Ziehl-Neelsen staining
 Mostly used to stain Mycobacterium tuberculosis that has
thick waxy cell walls with high content mycolic acids
(impermeable to dyes)
 Used to dived bacteria into two groups:
Acid Fast positive - appear red
Acid Fast negative - appear blue

Acid-Fast staining reagents :

1) Primary stain - Carbol Fuchsin + HEAT
2) Decolorizer - Acid alcohol
3) Counter stain - Methylene Blue
Acid-Fast staining procedure :
1) Flood the fixed smear with Carbol fuchsin
2) Heat to steaming for 5 min (do NOT boil or allow to dry &
replenish the evaporated stain), allow slide to cool & rinse
thoroughly with distilled water. ( heating melts (open) the
waxy cell wall of Acid-fast bacteria facilitating the
penetration of carbol fuchsin to get inside )
3) Flood the slide with 3% acid alcohol for 10 sec
4) Flood the slide with methylene blue, allow to stand for 1
min
5) blot dry & examine under Oil Immersion
 Differential – Endospore staining :
 AKA: Schaeffer-Fulton staining
 Endospores: resting (dormant) structure forming by G+ve
bacteria (Bacillus, Clostridium) in hard conditions
Endospore - appear green
Vegetative cell - appear red/pink
Endospores staining reagents :
1. Primary stain - Malachite green + HEAT
2. Decolorizer - Tap water
3. Counter stain - Safranin
Endospore staining procedure :
1) Flood the fixed smear with Malachite green over a
blotting paper
2) heat to steaming for 5 min (do NOT boil or allow to dry &
replenish the evaporated stain)
3) Tap water works as a decolorizer washing away the color
of the vegetative cell leaving it colorless While it can’t
wash away the color from inside the closed endospore
cell wall leaving it green
4) Flood the slide with Safranin, allow to stand for 1 min
5) blot dry & examine under Oil Immersion