Pharmaceutical Microbiology and Parasitology Laboratory

Common Laboratory Equipment Steps in media preparation:

ANALYTICAL BALANCE - The measuring pan is inside a  Weigh

transparent enclosure with doors so that dust does not  Dissolve
collect and so any air currents in the room do not affect the  Sterilize
balance's operation.  Dispense

AUTOCLAVE - A pressurized chamber used for the process STEP 1: WEIGH

of sterilization and disinfection by combining the three
factors: TIME, PRESSURE, STEAM  Using the weighing bioat start by weighing the
appropriate amount of agar for your batch into the
BUNSEN BURNER/ ALCOHOL LAMP - It is commonly weigh boat
used for sterilization.  The grams per liter will vary depending on the final
volume that you want and the culture media you are
HOT PLATE - Used to heat glassware and their components preparing
HOT air oven - Electric device that is used for sterilization of Example: HOW TO WEIGH THE APPROPRIATE AMOUNT
medical equipment or samples using dry heat. 100-110

INCUBATOR - Used in the laboratories for the growth and

maintenance of microorganisms and cultures

BIOSAFETY CABINET - Used to conduct process that are

sensitive to contamination

MAGNETIC STIRRER - Used for mixing various liquid

components.

INOCULATING LOOPS/NEEDLES

- Also known as smear loop, wire loop, inculation STEP 2: DISSOLVE

wand or microstreaker.
- Used to pick up and transfer small sample  Transfer the weighed agar into a clean Erlenmeyer
(inoculum) from a culture of microorganism. flask
 Add the appropriate amount of distiled water. Use
Petridish - Used to culture living cells such as bacteria and the water to wash down the sides of flask if some
fungi powder clingging to the flask
 Place the flask on a hot plate. Bring the agar to a
Test tube - Used in preparing media in slant/butt slant/butt boil while stirring using the stirring rod.
orientation  Be sure to mix and dissolve the agar completely so
there are no clumps.
Erlenmeyer flask - Used for preparing culture media
 Boiling for a longer period of time will turn the agar
Pipettes - used to transport specific amounts of fluids from dark and damage the nutritional properties of the
one place to another. media
 Cover the flask with cotton and foil
Beakers - Simple containers used to hold samples and
reagents STEP 3: STERILIZE

Stirring Rods - Used to mix solvents and samples together  Sterilize the agar by using autoclave
 Set the autoclave to 15 psi for 15 minutes at 121
Graduated cylinder - Cylindrical vessel that has volumetric degree celcius
markings
STEP 4: DISPENSE
MICROSCOPE - They are primarily used for the observation
of particles which cannot be observed with naked eye  Disinfect your working area before dispensing to
avoid contamination
CULTURE MEDIA PREPARATION  Prepare your sterile petri dish or slide
 Aseptically dispense approximately 25mL of agar in
Materials needed:
sterile petri plates
 Erlenmeyer flask  Ideally do this in front of HEPA filter if not available
 Stirring rod do the pouring in a room with minimal air and set up
 Foil your alcohol lamp to avoid contamination
 Agar
 Distilled water
 Hot plate with wire gauze
 Cotton
 Sterile petri dish and tubes
 Alcohol lamp
 Lighter
 Pharmaceutical Microbiology and Parasitology Laboratory
STAINING TECHNIQUES PRE-ANALYTICAL PHASE
- Giving color to give life  Sterilization of working area is very necessary by
using Lysol or bleach to wipe the table tops.
2 kinds of bacteria:  The laboratory scientist should always ware
 Gram positive bacteria – pink PERSONAL PROTECTIVE EQUIPMENTS at all
 Gram negative bacteria – purple / blue times inside the laboratory and considered all
specimens infectious.
2 shape bacteria:
 Cocci – has thick cell wall, gram positive bacteria, ANALYTICAL PHASE
round shape
 Bacilli – thin cell wall, gram negative bacteria, rod Materials;
shape  Broth cultures
 Slides
Kinds of Ionizable Dyes Used in Staining Bacteria  Inoculating loops
 Crystal Violet
BASIC DYES  Gram's iodine
 Cationic dyes with positively charged groups that  95% alcohol
adhere to negatively charge molecules like nucleic  Safranin
acids and proteins.
 Sticks in negative charge Procedure;
 Colored by examples: Nucleic acid, protein, celluole 1. Prepare bacterial smears from the bacterial
 Example: Methylene blue, crystal violet, safranin suspensions given.
and malachite green 2. Flood the smear crystal violet for one minute.
3. Wash off excess stain gently with running water.
ACIDIC DYES 4. Flood the smear with Gram's Iodine for one minute.
 Anionic dyes with negatively charged groups that 5. Wash off the Gram's Iodine with running water.
bind to positively charge cell structures. 6. Decolorize the smear by flooding with 95% alcohol.
 Sticks in positively charge Allow it to stand for 15-30 seconds. Repeat this
 Example: Eosin and acid fuchsin procedure until no more color comes off withthe
alcohol.
Cryptococcus staining – fungi that cause disease 7. Wash again with running water.
Chlamydia – obligate intracellular organisms 8. Counter-stain with Safranin for 30 seconds
Spiro – can see in dark field microscope/fluorescent 9. Wash off the excess stain with running water. Air
microscope dry.
10. Examine the stained smear under oil immersion
3 TYPES OF STAINING objective (OIO).
1. SIMPLE STAINING
 Single stain is used 4 REAGENTS IN GRAM STAINING
 Directed towards coloring the forms and shape of Gram staining Gram positive Gram negative
the cells step
 Example: Methylene blue Crystal Violet Blue/Purple Blue/Purple
Iodine Gram Blue/Purple Blue/Purple
2. DIFFERENTIAL STAINING Alcohol Blue/Purple Colorless
 Divide bacteria into separate groups safranin Blue/Purple Pink
 Directed towards coloring the components of the
elements present.
 Example: Gram staining and Acid-fast bacilli (AFB) GENERAL RULE:
staining
ALL COCCI ARE GRAM POSITIVE EXCEPT: (NeVerMind )
4 reagent:  Neisseria
 Application of primary stain  Veilonella
 Application of mordant  Moraxella/Branhamella
 Application of decolorizer
 Application of secondary stain aka counter staining ALL BACILLI ARE GRAM NEGATIVE EXCEPT:
 Arcanobacterium
3. NEGATIVE STAINING  Listeria
 Demonstrate presence of diffuse capsule  Bacillus
surrounding some bacteria  Mycobacterium
 Reagent used – black dye  Clostridium
 Example: India Ink or Nigrosin dye  Nocardia
 Cryptococcus neoformans – fungi that causes  Corynebacterium
diseases  Streptomyces
 Erysipelothrix
GRAM STAINING  Trophyrema whipplei
 CRYSTAL VIOLET --- PRIMARY STAIN – stain is
bluish purple GRAM (+) becomes GRAM(-)
GRAM’S IODINE --- MORDANT – enhances the  Over-decolorization
color of crystal violet  Old dying Acidic iodine
ALCOHOL --- DECOLORIZER – remove the color
SAFRANIN --- SECONDARY STAIN/COUNTER GRAM (-) becomes GRAM(+)
STAIN – stain in pinkish orange  Under-decolorization
 Thick smear
 Pharmaceutical Microbiology and Parasitology Laboratory

EXCEPTION:
 Organisms that exists almost exclusively within host
cells (Chlamydia)
 Organisms that lack cell walls (Mycoplasma and
Ureaplasma)
 Organisms with insufficient dimension to be
resolved by light microscopy (Spirochetes).

POST-ANALYTICAL
 After the experiment, all the glassware, materials
that have been contact with bacteria like inoculating
wire and including the working station, should be
sterilized properly. The student must prepare 10%
bleach or Lysol for disinfection materials like
inoculating wire can also be sterilized by flame. All
disposable materials should be place properly in
correct trash bags.
 PPE should be disposed in infectious trash bag,
and should not be worn outside the laboratory

SPECIAL STAINING METHODS

 Dyar – cell wall
 Anthony’s Hiss and Gin’s – capsule
 Nigrosin – capsule
 Neisser – matachromatic granules
 Albert – metachromatic granules
 Domer – endospore
 Schaeffer-Fulton – endospore
 Gray – flagella
 Leifson
 Feulgen – DNA
 Levaditi’s – Sphirochetes
 Fontana-Tribundeau – Spirochetes