CLINICAL BACTERIOLOGY|LABORATORY

PRELIM LESSON : MICROSCOPY & STAINING

TOPIC OUTLINE STAINING METHODS
 Introduction and Historical Background • POSITIVE STAINING – actual cells are stained and appear in a clear
1. Microscopy background.
2. Staining Methods ▪ Simple Stain – a stain which provides color contrast but gives the same
2.1. Gram Staining color to all bacteria and cells.
2.2. Acid-Fast Staining o Example: Loeffler’s Methylene Blue Staining
3. Reporting for Direct Sputum Smear Microscopy (NICE-TO-KNOW) ▪ Differential Stain – a stain which imparts different colors to different
MICROSCOPY bacteria and cells.
• Microscope’s main function is to magnify objects and its magnification depends o Example: Gram Staining, Acid-Fast Staining
on the type of utilized objectives and the ocular lens. • NEGATIVE STAINING – the background is colored to create a contrast to aid in
▪ Antonie Van Leeuwenhoek – a Dutch optician credited as the inventor of the better visualization of cellular structures.
microscope. - Uses dark dyes.
• Initial Magnification (IM) = Objective Lenses’ Magnification o Example: India Ink Staining
• Total Magnification (TM) = Ocular Eyepiece Magnification × Initial Magnification GRAM STAINING
▪ Eyepiece magnification = 10x • Developed by Hans Christian Gram in 1884.
▪ When asked about the MAGNIFICATION or the WORKING OBJECTIVE • COCCI – Round Shape
LENS, give just the INITIAL MAGNIFICATION. • BACILLI – Rod Shape
▪ When asked about TOTAL MAGNIFICATION WHEN USED, give the • Gram Positive – Purple/Violet (thick peptidoglycan layer)
TOTAL MAGNIFICATION • Gram Negative – Red/Pink (thin peptidoglycan layer)
OBJECTIVE LENS I.M. T.M. PURPOSE • All COCCI are Gram Positive (+) except N-V-M
SCANNER 4x 40x For scanning
▪ Neisseria spp.
LOW-POWER OBJECTIVE LENS 10x 100x For low power magnification ▪ Veilonella spp.
HIGH-POWER OBJECTIVE LENS 40x 400x For high power magnification ▪ Moraxella spp. (also: Branhamella)
OIL IMMERSION OBJECTIVE LENS 100x 1000x For increased resolution • All BACILLI are Gram Negative (-), except:
ADDITIONAL NOTES: CATALASE ACTIVITY
• Compound microscope is one of the most basic yet most important apparatus Listeria
in Microbiology. Catalase (+)
Bacillus
• IRIS DIAPHRAGM VS CONDENSER LBC has POSITIVE reviews
Corynebacterium
▪ Iris Diaphragm – controls the amount of light that passes through the Clostridium
aperture. With special characteristics
Actinomyces
o Can be adjusted through the protruding lever. They CAN because they are special
Nocardia
▪ Condenser – controls and adjusts the contrast of light. Mycobacterium Catalase (+)
• Rheostat – light adjustment knob Erysipelothrix
• Stage Adjustment Knob – controls forward/reverse and side to side movement Catalase (-)
Lactobacillus
of the stage NEGAVITE governance leads to WELGA.
Gardnellla
▪ Top knob – forward/reverse; back/forth; front to back M(+)
Arcanobacterium
▪ Bottom knob – side to side; left to right

STEPS: PRIMARY STAIN → RINSE → MORDANT → RINSE → DECOLORIZER → RINSE → COUNTER STAIN→ RINSE CELL COLOUR
APPLICATION REAGENT TIME COMPOSITION GRAM + GRAM –
Primary Stain Crystal Violet 30s 10g of 90% CV + 500mL Absolute Methyl Alcohol Violet Violet
Mordant (Seal) Gram’s Iodine 30s 6g Iodine Crystal + 12g Potassium Iodide + 800mL Distilled Water Violet Violet
Decolorizer Ethyl Alcohol 10s 400mL Acetone + 200mL of 95% Ethyl Alcohol Violet Colorless
Counter Stain Safranin 30s 10g of 99% Safranin + 1L Distilled Water Violet Red

ACID-FAST STAINING ADDITIONAL NOTES:

• Mycobacterium – Acid Fast Bacilli • MORDANT VS ACCENTUATOR
• Nocardia – Slightly Acid Fast Bacilli ▪ Mordant – serves as the link or bridge between the tissue (cell wall) and
• Other spp: Non Acid Fast the dye to make the staining reaction possible.
• Head Method – Ziehl-Neelsen ▪ Accentuator (Catalase) – is not essential to the chemical union of the
• Cold Method – Kinyoun’s tissue and the dye but accelerates the reaction.
• Acid Fast Bacilli – Red • Acid Fast Bacilli have strong mycolic acid
• Non-Acid Fast Bacilli – Blue • If the species is NOT Mycobacterium or Nocardia, then it is NON ACID FAST
• HCl (Hydrochloric Acid) makes Acid Alcohol acidic

STEPS: PRIMARY STAIN → MORDANT → RINSE → DECOLORIZER → RINSE → COUNTER STAIN→ RINSE CELL COLOUR
TIME NON-
APPLICATION REAGENT COMPOSITION AFB
ACADEME WORK AFB
(0.3g Basic Fuchsin + 10mL Ethanol) + (100mL of 5%
Primary Stain Carbol Fuchsin Flood w/ CF 30s Red Red
Phenol Solution in Distilled Water)
Ziehl-Neelsen – Heat until white Heat a white
Mordant Heat smoke appears smoke appears Red Red
(Seal) Kinyoun’s - Tergitol 30s 30s
Decolorizer Acid Alcohol 60s 10s 3mL concentrated HCl + 97mL Ethanol Red Colorless
Counter Stain Methylene Blue 60s 30s 0.3g Methylene Blue + 100mL Distilled Water Red Blue
BSMLS | MMLS 3-5 (2023) | 1
CLINICAL BACTERIOLOGY|LABORATORY
PRELIM LESSON : MICROSCOPY & STAINING
REPORTING FOR DIRECCT SPUTUM SMEAR MICROSCOPY
0 No AFB seen in 300 visual fields Negative
+n 1-9 AFB seen in 100 visual fields Questionable (Repeat Testing)
1+ 10-99 AFB seen in 100 visual fields
2+ 1-10 AFB/OIF in at least 50 visual fields Clinically Significant
3+ >10 AFB/OIF in at least 20 visual fields

EXAM TIPS:
• Una lagi tignan yung shape; bacilli or cocci, then LBC-CAN-MELGA
• Take note of the NOMENCLATURE, proper way of writing in print or in script
• Aralin yung composition kasi lagi daw lalabas kahit sa boards
• Para hindi malito sinong mauuuna kay Clostridium at Corynebacterium sa LBC-
CAN-MELGA, clostridium tetanus is pwedeng makuha kapag nasugatan ng lata
or CAN.

BSMLS | MMLS 3-5 (2023) | 2