Microscopic > End result is Red

SEM
20 nm
Endospores stain
Compound Microscope Primary Stain: Malachite green
0.2 um Mordant: Heat (steam)
Decolorizing Agent: Water
Human eye Counterstain: Safranin
0.2 mm > help bacteria survive harsh conditions like
heat, chemicals, and drying out
Motility
- Able to swim Capsule Stain
- Peritrichous Primary Stain: Crystal violet or India ink
(also known as nigrosin)
Non-Motility Mordant: None (typically not used)
- Non able to swim Decolorizing Agent: None (typically not used)
Counterstain: Safranin
> them appear as clear halos against a dark
4 Kinds of staining background
> the safranin sed as a counterstain to color
Simple Staining the background, enhancing the visibility of
- Crystal violet the capsules

Differential Staining Benedict

- Gram staining, Primary Stain: Crystal violet
- Acid fast stain Mordant: Gram's iodine (Potassium iodide and
- Endospores Iodine)
Decolorizing Agent: Ethanol or Ethyl alcohol
Selective Staining Counterstain: Safranin
- Acid-fast staining for detecting Mycobacterium Uses:
tuberculosis. > presence of reducing sugars in a
substance
Special Staining
- Flagella staining Gimenez stain (highlight them against a
- capsule stain green background)
Primary Stain: Fuchsin specifically basic
Staining Process fuchsin
Counterstain: Brilliant green or Light green
a) Flagella Staining Decolorizing Agent: Acid alcohol
Primary Stain: Carbolfuchsin or Basic Uses:
fuchsin > This helps identify bacteria like Rickettsia
Mordant: Tannic acid or Potassium alum and Chlamydia.
Decolorizing Agent: 95% alcohol or
Acetone Schaeffer-Fulton Stain (identify bacterial
Counterstain: Light green or Nigrosin endospores)
Primary Stain: Basic fuchsin
Nigrosine Mordant: Heat (steam)
- is For Negative Decolorizing Agent: Acid-alcohol
- India Ink Counterstain: Methylene blue

b). Congo red capsule stain (background Fluorescent Method

blue) - Staining technique that uses fluorescent dyes
Primary Stain: Congo red molecules or structures for visualization under a
Mordant: None fluorescent microscope.
Decolorizing Agent: Acid alcohol
Counterstain: Acid fuchsin Acid-Fast Staining (AFM):
Primary Stain: Carbol fuchsin
Mordant: Heat (steam) tsi (Triple Sugar Iron)
Decolorizing Agent: Acid-alcohol (9%) “Aim to Diff among bet members of
Counterstain: Methylene blue Enterobacteraceue family
K (Red) to A (Yellow)
Albert stain method (AFM): > Yellow = Acid
Primary Stain: Carbol fuchsin > Yellow in slant = Lactose
Mordant: None (heat is used instead of a > Yellow in Butt = Sucrose, Glucose
separate mordant) A/A = E.coli
Decolorizing Agent: Acid-alcohol K/K Red
Counterstain: Methylene blue Red/Yellow (Butt)
> helps to differentiate these bacteria from
others present in tissue samples > K = Lactose fermenting (Yellow)
> A = (Red)

Anion (Negatively charged Ion) BUTT K

- Nigrosine = India Ink Staunt A
Oos A-
Unstained Dye H2s Check (black
- Dark Field precipitate)
- seen against a dark background, making it
stand out more clearly LIA
> LDC (+) purple ‘slant’ “due of alkaline”
Phase Contrast > LDA (+) red
- observe live organism > Glucose (LDA + & H2S+) ”only fermented”
= yellow
Acid Fast Staining (AFM) > H2s – black precipitate
- 9% decolorizer
1.) Indole (Indole production
What is the color of the GRAM POSITIVE 2.) Sim (Sulfur production, motility)
specimen after decolorizer? Sulfide Production =
- specimens retain the purple color of the crystal Indole Production = Product Tryptophan
violet stain. breakdown (Product = kovacs)
Black (+) If theres indone
Non-Black (-) if none

What color is a NON ACID FAST specimen Motility =

after decolorizer? Turbid (Motil)
- colorless NonTurbide (Non motile)

What is methylene blue in AFM
A. Primary dye B. Secondary Dye C. Tertiary
Dye D. Quaternary Dye
Lowenstein
- medium is a specialized growth medium used
for culturing mycobacteria,
What is the color of the GRAM POSITIVE
specimen after decolorizer?
- specimens retain the purple color of the crystal
violet stain.
When theres oxygen it will have TCH
Black (Hydrogen Sulfie produce)
Methyl Red Test (mrt) “3 drops”
Red colour = Acid produce ‘strong’ (+)
Yellow color = weak acid or acetoin (-)
vpt test
citric test
- determine microbes to ferment
> Blue (+) indicate sodium
> Green (-)
Ecoli (-) , Chla (+)

urease test
BIOCHEMICAL TEST > pink (+)
> yellow (-)  Klebsiella: LDC (+),
Ecoli (-), Chla (+) LDA (+), H2S (-)
3. Indole Test
 Aim: Detect indole
1. TSI (Triple Sugar Iron) production
 Aim: Differentiate among  Interpretation:
members of  Indole Production:
Enterobacteriaceae family  Product of
 Interpretation: Tryptophan
 K (Red) to A (Yellow) breakdown
 Yellow:  Kovacs
 Acid Reagent:
 Yellow in  Black
slant: (+) if
 Lactos indole
e  Non-
 Yellow in Butt: Black
 Sucros (-) if
e, none
Glucos  E. coli: Indole (+)
e  Klebsiella: Indole (-)
 A/A:
 E. coli (A/A) 4. SIM (Sulfide Indole Motility)
 K/K:
 Lactose  Aim: Test for sulfur
fermenting production, indole
(Yellow) production, and motility
 Red/Yellow (Butt)  Interpretation:
 H2S Check:  Sulfide Production:
 Black  Black (+) for
precipitate sulfur
2. LIA (Lysine Iron Agar)

 Aim: Differentiate
Enterobacteriaceae based on  Indole Production:
lysine and glucose  Kovacs
fermentation Reagent:
 Interpretation:  Black
 LDC (+) purple "slant" (+) if
 LDA (+) red indole
 Glucose (LDA + &  Non-
H2S +): Black
 Only (-) if
fermented = none
yellow  Motility:
 H2S:  Turbid (Motile)
 Black  Non-Turbid
precipitate (Non-motile)
 E. coli: LDC (-), LDA  E. coli: Motile (+)
(-), H2S (-)  Klebsiella: Motile (-)
5. Methyl Red Test (MRT)  Non-urease-
producing
 Aim: Detect mixed acid  E. coli: Urease (-)
fermentation  Klebsiella: Urease
 Interpretation: (+)
 Red color:
 Strong Acid
Production (+)
 Yellow color:
 Weak Acid or
Acetoin (-)
 E. coli: MRT (+)
produce strong acid
 Klebsiella: MRT (-)

6. VP (Voges-Proskauer) Test

 Aim: Detect acetoin

production
 Interpretation:
 Acetoin Production:
 Red (+) after
Barritt's
Reagent A & B
 Yellow (-)
 E. coli: VP (-)
 Klebsiella: VP (+)

7. Citrate Test
 Aim: Determine citrate
utilization
 Interpretation:
 Blue (+):
 Citrate
utilization
 Green (-):
 No citrate
utilization
 E. coli: Citrate (-)
 Klebsiella: Citrate (+)

8. Urease Test

 Aim: Detect urease

production
 Interpretation:
 Pink (+):
 Urease-
producing
 Yellow (-):