ROBIN'S GUIDE TO MICROSCOPE CREATION

The information below is meant as a guide to creating "microbiologically correct" microscope slides at DU. Use this information in choosing your microscope type and staining/preparation procedures.

Type of Microscope	Stain/Slide Preparation	What you see
Brightfield Microscope purpose: to illuminate the object being viewed from alight source below the field of focus	a) Gram's Stain b) Simple Stain (methylene blue) c)Wright's stain	bright background; colored specimen
Darkfield Microscope purpose: to allow light to be reflected off the surface of the object; used to view living organisms	a) wet mount (living) b) heat fixed (dead)	specimen is bright against a dark, black background
Phase-Contrast Microscope purpose: for direct observation of unstained material; used to observe living organisms	a) wet mount - direct b) hanging drop c) direct mount - saline or iodine	specimen is highlighted, both bright and dark depending upon density; living cells can be used
Fluorescence Microscope purpose: used to visualize cells that have become illuminated by fluorochromes	a) acridine orange b) rhodamine-auramine acid fast c) calcofluor white d) acid fast	specimens appear brightly lite against a dark background; color of light depends upon fluorochrome used
Scanning Electron Microscope purpose: useful in observing surface details, via scanning beams of electrons	a) carbon coating b) metal coating	a 3-D view of bacteria in black and white; you observe surface structures, not internal ones

Stain Method/Slide Preparation	Purpose
Gram's Stain	commonly used to detect the difference between bacteria that retain crystal violet (Gram +) and those that cannot and stain red (Gram -)
Methylene Blue (simple stain)	used to show microbes and bacteria in direct smears; gross morphology only (shape and arrangement)
Hanging Drop	used for studying motility of bacteria - less distortion from weight of cover slip and deeper field of focus
Flourescent dyes (auramine and rhodamine)	reacts directly with cell wall of the Mycobacteria, causing specimen to illuminate with the use of UV light; can be used with antibodies to direct staining
Saline wet mount	useful for detection of helminth eggs or larvae and refractile protozoan cysts; iodine enhances nuclear detail and glycogen masses
Calcofluor White	used for the detection of fungi
Acridine Orange	binds to nucleic acid; stains the nuclear area of prokaryotic cells
Direct Wet Mount	parasites are best visualized by this type of mount

Stain	Description
Gram's Stain	commonly used for primary microscopic examinations of specimens submitted for smear or culture; suited for specimens that strongly suspect bacterial infections Gram positive bacteria stain dark blue or blue-black; all other elements stain a safranin red.
Acid Fast Stain	used to detect mycobacteria in specimens The primary stain binds to mycolic acid in the cell walls of the mycobacteria and is retained with decolorization with acid alcohol. Variations on acid fast techniques include: a) fluorescent, b) Kinyoun, and c) Ziehl-Neelsen Appearance: a) mycobacteria stain bright orange or yellow-green depending upon fluorochrome used, b and c) mycobacteria stain red with background and non-acid fast organisms staining blue
Calcofluor White Stain	Calcofluor white is a colorless dye that binds to cellulose and chitin and becomes flourescent with long and short wavelength UV light. Used for rapid screening of clinical specimens for fungal elements Yeast cells, pseudohyphae and hyphae display a bright apple green or blue/white fluorescence Special filters/light source and microscope required.
Wright- Giemsa Stain (rapid modified)	neutral dye of thiazinc and acid eosin attach to oppositely charged sites on proteins It is a rapid stain for smears and imprints to fully stain background materials and cells and a wide variety of microorganisms.
Methylene Blue Stain (simple stain)	commonly used to observe shape and arrangement of bacteria used to stain Corynebacterium diphtheriae for observation of metachromatic granules also used as a counter stain in the Kinyoun and Ziehl-Neelsen acid fast staining procedures

Bacteria/Microorganism	Suggested Stain/Slide Preparation
Staphylococcus species	gram's stain
Streptococcus species	gram's stain
Mycobacterium tuberculosis	acid fast stain
Campylobacter species	direct wet mount
Borrelia burgdorferi	silver stain/acridine orange stain
Pneumocystis carinii	calcofluor white stain
Chlamydia species	gimenez stain
Legionella species	gimenez stain
Corynebacterium diphtheriae	methylene blue simple stain
Cryptosporidium	acid fast stain

Specimen	Suspected Disease	Lab Procedure
throat culture	diphtheria	gram's stain methylene blue simple stain
sputum	bacterial pneumonia	gram's stain
sputum	tuberculosis	acid fast stain
wound	bacterial cellulitis	gram's stain
cerebrospinal fluid	bacterial meningitis	gram's stain methylene blue simple stain acridine orange stain
cerebrospinal fluid	listeriosis	gram's stain hanging drop wet mount
urine	yeast infection	calcofluor white gram's stain
urine	bacterial infection	gram's stain
feces	cholera	direct mount - alkaline prep
feces	parasitic disease	direct mount - saline or iodine