Staining

Gram Stain
Principle:
Classify bacteria as Gram positive or Gram
negative

Reagents:
Crystal Violet
95% Ethyl Alcohol
Grams Iodine
Safranin
Water

Gram Staining
(Microorganisms)
Gram Positive
(PURPLE)
Staphylococcus
epidermis (24)
Shape: Cocci (round)
Arrangement:
Staphylococcus (cluster)

Bacillus cereus (03)

Shape: Bacilli
Arrangement:
Streptobacilli (long chain)

Gram Negative
(RED)
Escherichia coli (11)
Shape: Bacilli
(rod)
Arrangement:
Diplobacillus (2
rods)

Acid-Fast Stain
Principle:
Classify acid-fast & non-acid fast

Reagents:
Carbol fuchsin
5% Acid Alcohol
Methylene Blue
Water

Acid-Fast (Microorganism)
Acid-Fast
(MAGENTA)
Mycobacterium
smegmatis (16)
Shape: Bacilli
(rod)
Arrangement:
Pleomorphic

Non Acid-Fast
(BLUE)
Micrococcus luteus
(15)
Shape: Cocci
(round)
Arrangement:
tetrad

Endospore Staining
Principle:
Difference between endospore &
vegetation

Reagent:
Malachite Green
Water
Safranin

Endospores (Microorganism)
Endospore Producers
Bacillus cereus (03)
Vegetative cell (RED)
Shape: Bacilli
Arrangement: Streptobacilli

Endospore (GREEN)

Clostridium botulinum

Capsule Staining
Reagents:
1% Crystal violet
Nigrosin

Microorganisms
Klebsiella pneumoniae (13)
Shape: Bacilli
Color of bacteria: PINK/PURPLE
Color of capsule: Clear

Fungal Slides

Rhizopus stolonifer (bread

mold)

Zygospore
(Sexual Spores)

Rhizopus stolonifer (bread

mold)

Sporangium sac & Sporangiospores

(Asexual Spores)

Aspergillus niger

Conidiospores
(Asexual Spores)

Penicillium notatum

Conidiospores
(Asexual Spores)

Corprinus sp. (Mushroom)

Basidiospores
(Sexual Spore)

Cryptococcus neoformans

Yeast
(Budding)

Parasitic Protozoan Slides

Trypanosoma gambiense

Class: Euglenozoa
Mode of locomotion: flagellum
Mode of transmission: Tse Tse fly
(vector)
Disease: African sleeping sickness
***unable to make cyst***

Toxoplasma gondii

Class: Apicomplexa
Mode of locomotion: none
Mode of transmission: ingestion of
contaminated water, food, milk or inhalation or
inoculation
Disease: Toxoplasmosis
(Trophozoite only)

Balantidium coli
Trophozoite

Cyst

Class: Ciliates
Mode of locomotion: cilia
Mode of transmission:
ingestion of contaminated
water or food

Giardia lamblia
Trophozoite

Cyst

Class: Euglenozoa
Mode of locomotion:
flagellum
Mode of transmission: cyst

Plasmodium vivax
Class: Apicomplexa
Mode of locomotion: none
Mode of transmission: Mosquito
(anophele)
Disease: Malaria

Merozoite

Plasmodium vivax
(continued)
Signet
ring

Plasmodium vivax
(continued)
Trophozoi
te

Early
Schizont

Plasmodium vivax
(continued)
Late
schizont

Entamoeba histolytica

Class: Amebae
Mode of locomotion:
pseudopodia
Mode of transmission:
ingestion of contaminated

Parasitic Helminthes

Tania pisiformis (dog tapeworm)

Phylum: Platyhelminthes
Class: Cestoda
Monoeicious
Mode of transmission:
ingestion of undercooked
beef or pork

Tania pisiformis (dog

tapeworm)
Hooks

Scolex

Sucker

Neck

Proglottids

Ascaris lumbricoides
Male

Female

Phylum: Aschelminthes/
Nemathehelminthes
Class: Nematoda
Diecious
Mode of transmission: ingestion of
feces contaminated food or soil
Treatment: mebendazole or pryantel

Fasciola hepatica (liver fluke)

Phylum: Platyhelminthes
Class: Trematoda
Monoeicious
Mode of transmission: ingestion of
poorly cooked fish, crab, crayfish and
aquatic plants
Treatment: praziquantel (Biltricide)

Enterobius Vermicularis (pinworm)

Female

Phylum: Aschelminthes
Class: Nematoda
Mode of transmission:
ingestion of eggs
Treatment: Mebendazole or
pryantel pamoate

Schistosoma japonicum (blood

fluke)

Phylum: Platyhelminthes
Class: trematode
Diecious
Mode of transmission: contact with
snail infected water
Treatment: praziquantel

Necator americanus (hookworm)

Phylum: Aschelminthes
Class: Nematoda
Diecious
Mode of transmission: contact
with larvae infected vegetation or

Biochemial Test

Differential:
Blood/RBC

Blood Agar

Enterococcus faecalis
(10)
Gamma hemolysis
(no zone):
No hemolysis of
hemoglobin

Streptococcus pyogenes (29)

Beta hemolysis (clear zone):
Partial hemolysis of RBC &

Streptococcus
mitis (27)
Alpha hemolysis
(green zone):
Complete
hemolysis of
RBC &
hemoglobin

Selective: 7.5%
salt
Differential:
Mannitol

Mannitol Salt Agar

Escherichia coli
(11)
NONhalophile
(Bacteria
does not
grow)

Staphylococcus aureus (23)

Halophile (bacteria grows)
Ferments Mannitol ( & makes
acid= yellow growth)

Streptococcus
mitis (27)
NON-halophile

Staphyloccocus
epidermis (24)
Halophile
DOES NOT ferment

Staphylococcus
aureus (23)
G+ (DOES NOT grow)

MacConkey Agar

Enterobacter
aerogenes (09)
G- ( bacteria
grows)
Ferments Lactose
(little amount of
acid)

Escherichia coli
(11)
G Ferments
Lactose (&
makes large
amount of

Selective: GDifferential:
Lactose

Salmonella
typhimurium
(20)
G DOES NOT
ferment
lactose
(grayish)

Staphylococcus
aureus (23)
G+ (DOES NOT grow)

Selective: G+
Differential:
Lactose

Eosin Methylene-Blue Agar

Enterobacter
aerogenes (09)
G- ( bacteria grows)
Ferments Lactose
(weak fermenter=
pink-ish)

Salmonella
typhimurium (20)
G DOES NOT ferment
lactose (grayish)

Escherichia coli
(11)
G Ferments
Lactose (strong
fermenter=
green metallic)

Selective: G- (partial
growth)

Phenylethyl Alcohol Agar

(PEA)

Enterococcus faecalis
(10)
G+ (bacteria grows)
Escherichia coli
(11)
G- (bacteria
partial grows)
Staphylococcus
aureus (23)
G+ (bacteria grows)

Gas Pak Jar (Anaerobic

Conditions)

AEROBIC CONDITIONS

ANAEROBIC CONDITIONS

Micrococcus luteus(15):
Grows (Aerobic)

Micrococcus luteus(15)
Does not grow

Clostridium sporogenes(34)
Does not grow

Clostridium sporogenes(34)
Grows (Anaerobic)

Escherichia coli(11)
Grows best (Facultative
anaerobic)

Escherichia coli(11)
Grows very little

Gas Pak Jar

Methylene blue indicator:
Blue: Oxygen Present
White: No Oxygen Present

Gas Pak
Sodium borohydride & Sodium
bicarbonate

Gases Produced
Carbon Dioxide & Hydrogen (combines w/
free Oxygen to form Water)

Thioglycollate Broth Incubation

(Anaerobic Conditions)
Micrococcus luteus
(15)
DOES NOT grow
(Aerobic)
Clostridum
sporogenes (34)
Grows mainly in
the bottom
(Anaerobic)
Escherichia coli (11)
Grows ALL OVER (facultative
anaerobic)

UV Radiation

Can NOT be use for sterilization

G+ & G- are equally sensitive
Old cultures are not sensitive to UV radiation
(endospores)
Young cultures are sensitive to UV radiation

Antibiotics (Zone of
Inhibition)

Measure
diameter in
CM & convert
to MM
1 cm=10mm

Measure the radius from the

center of antibiotic to nearest
growth in
CM& convert to MM X 2

Antibiotic (Additive &

Synergistic)

Additive Effect
Penicillin & Tetracycline
Tetracycline &
Trimethoprim

Synergistic Effect
Penicillin &
Streptomyocin
Trimethoprim &

IMViC: Indole
Substrate: Tryptophan
Medium: SIM agar
Enzyme:
Tryptophanase
End-product: Indole
Reagent: Kovacs
reagent
Escherichia coli (11): RED -> Indole
Present (+)
Enterobacter faecalis (09): CLEAR ->
No Indole

IMViC: Methyl Red

Substrate: Glucose +
Peptone
Medium: MR-VP broth
Enzyme: N/A (many)
End-product: Stable Acids
Reagent: Methyl Red

Escherichia coli (11): RED -> Acid Present

(+)
Enterobacter faecalis (09): Yellow -> No
Acid Present (-)

IMViC: Voges-Prouskauer
Substrate: Glucose + Peptone
Medium: MR-VP broth
Enzyme: N/A (many)
End-product: Neutral Non-Acids
(acetonin)
Reagent: Barritts reagent A (alphanaphthol) Barritts reagent B
(potassium hydroxide)
Escherichia coli (11): Yellow -> Acid
Present (-)
Enterobacter faecalis (09): RED-> No
Acid Present (+)

IMViC: Citrate
Substrate: Citrate
Medium: Simmons Citrate
Agar
Enzyme: Citrate Permease or
Citrase
End-product: Sodium
bicarbonate
Indicator: Bromthymal Blue
Escherichia coli (11): GREEN-> Acidic (-)
Enterobacter faecalis (09): BLUE-> Alkaline/Sodium
bicarbonate (+)
Proteus vulgaris (17): BLUE-> Alkaline/Sodium
bicarbonate (+)

Carbohydrate Fermentation:
Substrate:
Media:
Phenol red lactose broth
Phenol red glucose broth
Phenol red sucrose broth
Enzyme:
End-product: Acid &
Ammonia
Indicator:
red
Glucose Phenol
Sucrose

Lactose

S. aureus (23)

Acid only

Acid only

Acid only

P. aeruginosa
(18)

Acid + gas

Acid + gas

E. coli (11)

Triple Sugar Iron

Purpose: to differentiate the
members of the Enterobariasie.
Determines if bacteria has made
Hydrogen Sulfide or gas, and the
sugars it ferments.
Substrate:
Acid: 1% sucrose, 1%,
lactose, 0.1% glucose
H2S: organic (cysteine and
cystene) & inorganic (sodium
thiosulfate)
Medium: TSI agar
Enzyme:
End-product: Acid, Hydrogen
Sulfide, Gas
H2S Indicator: Iron

Name

Slant/B
utt

Shigella dysenteriae
(22)

R/Y

Salmonella
typhimurium (20)

R/B

Psuedomonas
aeruginosa (18)

R/R

Proteus vulgaris (17)

Y/B

Escherichia coli (11)

Y/Y

H2S

Gas

Gluco
se

Sucro
se

lactos
e

Starch Hydrolysis
Substrate: Starch
Medium: Starch agar
Enzyme: Amylase
End-product: Maltose
Reagent: Grams Iodine

Amylase
E. Coli (11)

P. Aeruginosa
(18)

S. Aureus (23)

B. Cereus (03)

Yes

Casein Hydrolysis
Substrate: Casein
Medium: Milk agar
Enzyme: Caseinase
End-product: Amino acids
Reagent:

Caseinase

Halo

P. aeruginosa

yes

yes

S. aureus

yes

yes

B. cereus

yes

yes

E. coli

Gelatin Hydrolysis
Substrate: Gelatin
Medium: Gelatin
Enzyme: Gelatinase
End-product: Amino acids
Reagent:

Solid

Liquid

Gelatinas
e

yes

yes

yes

S. aureus

yes

B. cereus

yes

yes

E. coli
P.
aeruginosa

Nitrate Reduction Test

Substrate: Nitrate
Medium: Nitrate broth
Enzyme: Nitrate reductase
End-product: Nitrite, Ammonia
Reagent: Nitrate reagent A
(sulfanilic acid), Nitrate reagent B
(naphthylamine), Zinc powder

Name
A. Faecalis (01)
E. Coli (11)
P. Aeruginosa (18)

Reagent A & B

Zinc
powder

Endproduct

positive

positive

nitrite

ammonia

Nitrate Reduction Test

After adding Nitrate reagent A&B, if tube
turns:
RED (+) -> End product: Nitrite (E. coli 11)
Clear (-) -> Bacteria didnt break down to nitrate
or broke down into Ammonia (P. aeruginosa 18 &
A. faecalis 01)

Add Zinc powder, if tube turns:

CLEAR (+) -> Bacteria broke down into Ammonia/
Zinc didnt break down Nitrate (P. aeruginosa 18)
RED (-) -> Bacteria didnt break down/ Zinc
powder broke it down to Nitrite (A. faecalis 01)

Catalase Test
Substrate: H2O2
Media: Trypticase Soy Agar
Enzyme: Catalase, Oxidase,
Superoxide dismutase
End-product: H2O + O2
Reagent: Hydrogen peroxide
(H2O2)
Catal
ase

Foa
m

Micrococcus luteus
(15)

Staphylococcus
aureus (23)

Streptococcus lactis