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

Microorganisms were first observed by Antonie van ____________, using a ________ microscope. A _____________microscope has only one lens. Cowan “Microbiology”

RELATIVE SIZE as a TOOL in ________________ _____ System – International System of Units Cowan “Microbiology”

SOME TYPES of MICROSCOPES ________ Bright field __________ LIGHT MICROSCOPE _____________ ELECTRON Transmission Scanning

________________ MICROSCOPY DISTINGUISHING FEATURES Visible light passes through a series of ________ which magnify the specimen and allows fine detail to be observed (___________) Specimen appears _______ Field appears lighter PRINCIPLE USES Common, multi-purpose microscope Used to observe ____ specimens and preserved, stained (___-living) specimens Provides fair cellular detail

_________________ MICROSCOPE

___________ Light Microscopy PATHWAY of _____ In a compound microscope, the image from the ____________ lens is magnified again by the __________ lens Total magnification = objective lens  ocular lens Figure 3.1b

Properties of a Compound Light Microscope: 1. _____________________ Magnification of _________ Lens X Magnification of Ocular Lens = Total Magnification Low Power ___X 10X 100X High Dry 400X Oil Immersion 1000X 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)

_________ 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 in the 100x objective lens ____________ is used to keep light from bending Figure 3.3

_______________ MICROSCOPY DISTINGUISHING FEATURES Specimens are stained with ____________dyes ultraviolet light is used which causes ___________ molecules in a specimen to emit light PRINCIPLE USES Rapid detection and identification of organisms in tissues Excellent _____________ tool

ELECTRON MICROSCOPE TYPES: _____________________ ___________________________

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

___________ ELECTRON MICROSCOPY DISTINGUISHING FEATURES Beam of __________ (not light) pass through the specimen ___ dimensional image is produced Magnification 10,000 to 100,000x PRINCIPLE USES Examination of _______ _____________ of cells

COMPARISON of _____ MICROSCOPES and ____________ MICROSCOPES Cowan and Talaro

MICROSCOPIC OBSERVATION TWO TYPES of SPECIMENS: 1. ____________ Wet Preps, Wet Mounts. Living organisms suspended in fluid-organisms have ________________with surrounding fluid. Used to study: size, shape, arrangement of cells, (morphology), __________ , 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 motility.

PREPARATION of _______ SMEAR SMEAR: A ___ film of a solution of ________ on a slide A smear is usually ____ to ______ the microbes to the slide and to ______ the microbes. ______: Coloring the microbe with a ____ that emphasizes certain structures _________ STAINING Nester et al.

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

VARIOUS STAINING CATEGORIES cntd. SPECIAL Targets _______ cell _______ Such as: capsules flagella spores Ex.: India Ink Flagella Stain Spore Stain SIMPLE _____ dye Simple procedure Ex.: Methylene Blue Crystal Violet Safranin Malachite Green DIFFERENTIAL Two dyes __________ Contrast 2 cell types or parts Complex procedure Ex.: _____ Stain ___________ Stain

_______________ STAIN GRAM STAIN Developed by Dr. Hans Christian Gram in 1884 Most widely used procedure for staining ___________ Classifies bacteria into two groups Based on differences in _________ STRUCTURE Gram positive Gram negative

Basic _____ of Most Bacterial Cell _____ Basic _______ of Bacterial Cell Walls: determine _______ provide structural _________

COMPARISON of GRAM POSITIVE and GRAM NEGATIVE BACTERIA Ex.: Staphylococcus aureus Streptococcus pyogenes GRAM _________ BACTERIA Ex.: Escherichia coli Klebsiella pneumoniae Nester

Gram-positive bacterial cells Gram-negative bacterial cells Gram Stain PROCEDURE STEPS: Color of Gram-positive bacterial cells Gram-negative bacterial cells 1. ________ stain: Crystal Violet Purple 2. ____________: Iodine 3. _____________ agent:** Acetone Alcohol _________ 4. Counterstain: _____________ ___________ ________

_______ STAINING PROCEDURE Tortora

________________ STAIN Acid Fast Stain Used for bacteria with _______ material in cell wall Several procedures* Ex.: Mycobacteria species Nocardia species _____bacteria species Nester

DIFFERENTIAL STAIN ________________ Used for bacteria with waxy, lipid (_____ ______) material in cell wall Several procedures* 1. Primary stain: Carbolfuchsin 2. Decolorizer: _____ Alcohol 3. Counter stain: Methylene blue Ex.: Mycobacteria species Nocardia species Mycobacteria species Nester

___________ Stain Procedure STEPS: Color of ___________ Bacteria (Mycobacteria sp.) Non–Acid-fast Bacteria 1. Primary stain: __________ Red 2.__________ agent:** _____-alcohol ___________ 3. Counter-stain: _____________ ____ ______

ACID FAST STAIN Acid-fast staining of a patient’s ________ 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?

SPECIAL STAINS Used to distinguish _____ of cells CAPSULE ______________ ENDOSPORE Tortora

COMPARISON of STAINS Cowan et al.

SOME IMPORTANT STAINS USED in MICROSCOPY ________ - Methylene Blue - Carbolfuchsin - Crystal Violet - Safranin DIFFERENTIAL - Gram - Acid-Fast SPECIAL - __________ (Negative, Acidic) - Endospore - Flagella