CHAPTER 3 OBSERVING MICROORGANISMS THROUGH A MICROSCOPE Dr. Reitano SUFFOLK COUNTY COMMUNITY COLLEGE

Microorganisms were first observed by Antonie van Leeuwenhoek, using a simple microscope. A simple microscope has only ____ _____. Cowan “Microbiology”

_________ ____ as a TOOL in IDENTIFICATION Cowan “Microbiology”

THE __________ SYSTEM International System of Units Equals Note: millimeters are smaller than centimeters, but there are a larger number of millimeters in a given length.

SOME TYPES of MICROSCOPES LIGHT -uses visible light to observe specimen – Bright field COMPOUND LIGHT MICROSCOPE – Fluorescence ELECTRON-uses electrons instead of visible light – Transmission – Scanning

BRIGHT FIELD MICROSCOPY DISTINGUISHING FEATURES – ______ light passes through a series of lenses which ________ the specimen and allows fine detail to be observed (____________) – Specimen appears darker – Field appears lighter PRINCIPLE USES – Common, multi- purpose microscope – Used to observe live specimens and preserved, stained (non-living) specimens – Provides fair cellular detail

____________ LIGHT MICROSCOPE

Figure 3.1b Compound Light Microscopy In a compound microscope, the image from the _____________ is magnified again by the ocular lens Total magnification = objective lens  ocular lens PATHWAY of LIGHT

Properties of a Compound Light Microscope : 1. __________________ Magnification of Objective Lens X Magnification of Ocular Lens = Total Magnification Low Power10X 100X High Dry40X10X400X Oil Immersion 100X10X1000X 2. ______________ The ability of a lens system to accurately distinguish between two separate points, that lie close to each other, as separate and distinct. (structures less than 0.2um cannot be resolved with the compound light microscope)

Figure 3.3 __________ in the Compound Microscope _________ index - a measure of the light-bending ability of a medium, such as air Air may bend the light so much that it misses the small size of the opening of the 100x objective lens Immersion oil is used to keep light from bending

______________ MICROSCOPY DISTINGUISHING FEATURES – Specimens are stained with fluorescent dyes – Ultraviolet light is used which causes fluorescent molecules in a specimen to emit light PRINCIPLE USES Rapid detection and identification of organisms in tissues Excellent _________ tool

_____________ MICROSCOPE TYPES : _________________ TRANSMISSION

__________ ELECTRON MICROSCOPY DISTINGUISHING FEATURES Beam of electrons (not ______) are reflected from the specimen _______ dimensional image produced Magnification 1,000 to 10,000x Principle Uses Observing _______ details of cells and _________

___________ ELECTRON MICROSCOPY DISTINGUISHING FEATURES – Beam of electrons (not light) pass through the specimen – ____ dimensional image is produced – Magnification 10,000 to 100,000x PRINCIPLE USES PRINCIPLE USES Examination of viruses Examination of viruses ____________ of cells ____________ of cells

ELECTRON PHOTOMICROGRAPHS Tortora et al.

COMPARISON of ____ MICROSCOPES and ELECTRON MICROSCOPES Cowan and Talaro

MICROSCOPIC OBSERVATION TWO TYPES of SPECIMENS: – 1. ______________ Wet Preps, Wet __________. Living organisms suspended in fluid-organisms have little contrast with surrounding fluid. Used to study: size, shape, arrangement of cells, (morphology) behavior, and _______________. – 2. STAINED Fixed Smear Preparations. _____________organisms. Contrast is created to allow cellular characteristics to stand out. Used to study size, shape, arrangement of cells (morphology) but not _______________.

PREPARATION of ________ SMEAR _________ STAINING Nester et al. Smear: A thin film of a solution of ________ on a slide. A smear is usually fixed to ______ the microbes to the slide and to kill the microbes. Staining: ________ the microbe with a dye that emphasizes certain structures.

VARIOUS STAINING CATEGORIES _______ STAINS _________ charge (negative stain) Repelled by cells _________ is stained Ex.: India ink capsule stain BASIC STAINS __________ charge (positive stain) ________ to cells (which have a negative charge) Cells are stained Ex.: Methylene ______, Crystal Violet, Safranin, Malachite Green

VARIOUS STAINING CATEGORIES _________  ____ dye  Simple procedure  Ex.: Methylene Blue Crystal Violet Safranin Malachite Green ______________  ____ dyes  Primary  Counterstain  Contrast 2 cell types or parts  Complex procedure  Ex.:  _____ Stain  Acid-fast Stain SPECIAL  Targets specific cell parts  Such as: _________ flagella spores  Ex.:  India Ink  Flagella Stain  Spore Stain

DIFFERENTIAL STAIN _______ STAIN Developed by Dr. Hans Christian Gram in 1884 Most widely used procedure for staining bacteria Classify bacteria into two groups – Based on differences in CELL _____ STRUCTURE Gram ________ Gram negative

Basic Structure of Most _________ Cell ______ determine _____________ provide structural support Basic Function of Bacterial Cell Walls:

COMPARISON of GRAM POSITIVE and GRAM NEGATIVE BACTERIA GRAM _________ BACTERIA  Ex.: Staphylococcus ________ Streptococcus pyogenes GRAM ___________ BACTERIA  Ex.:  Escherichia ______  Klebsiella pneumoniae Nester

STEPS: Color of Gram-positive bacterial cells Color of Gram-negative bacterial cells 1. _______ stain: Crystal violet Purple__________ 2. __________: Iodine Purple 3. Decolorizing agent:** Alcohol-acetone PurpleColorless 4. _______stain: Safranin Purple______ GRAM STAIN PROCEDURE

GRAM STAINING PROCEDURE Tortora

DIFFERENTIAL STAIN ______________ Stain Used for bacteria with waxy, lipid (mycolic acid) material in cell wall Several procedures* 1. ________ stain: Carbolfuchsin 2. Decolorizer: ______ Alcohol 3. Counter stain: Methylene blue Ex.: – Mycobacterium species – Nocardia species Mycobacterium species Nester

STEPS: Color of Acid-fast Bacteria (Mycobacteria sp.) Color of Non–Acid- fast Bacteria 1. Primary stain: Carbolfuchsin Red 2. Decolorizing agent:** Acid-__________ Red___________ _ 3. Counterstain: Methylene _____ Red___________ ACID-FAST STAIN PROCEDURE

ACID FAST STAIN Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose ___________. What is the genus and species of this organism? This is an acid-fast stain of a patient’s __________. What is the disease associated with this organism?

__________ STAINS Used to distinguish _____ of cells CAPSULE FLAGELLA ENDOSPORE Tortora

COMPARISON of STAINS Cowan et al.

Identify the Types of Stains Used

SOME STAINS USED in MICROSCOPY SIMPLE - Methylene Blue - Carbolfuchsin - Crystal Violet - __________ DIFFERENTIAL - Gram - __________ SPECIAL - Capsule (Negative, Acidic) - __________ - Flagella