MICROLAB PREPARATIONS A) Lab Safety Rules Lab Safety Form Signup B) Lab Participation Instructor Review Peer Review Form C) Biohazard Rules How to dispose Trash REQUIRED Items: 1) LAB Manual/Journal 2) Scantrons 882-E, No.2 pencil USEFUL Items 1) Colored Pencils Purple/Pink/Red/Blue/Green BIOHAZARD Rules VIOLATORS will lose points C1) NO FOOD/DRINK ALLOWED in LAB C2) CORRECT TRASH in CORRECT CONTAINER Do not put regular trash in any of the biohazard bins All laboratory supplies (which touches live biospecimen) and biospecimen must be placed in the BIO HAZARD bin. C2A) BIOHAZARD Cardboard Container (plastic/paper) C2B) BIOHAZARD Plastic Container (glass culture vials) you will learn the basic skill sets necessary to Lab 1, 2 and 3: Stain, Observe and Identify the Microbes ACGM/CSLO Goals: 1. Use and comply with laboratory safety rules, procedures, and universal precautions. 2. Demonstrate proficient use of a compound light microscope. 3. Describe and prepare widely used stains and wet mounts, and discuss their significance in identification of microorganisms. 4. Perform basic microbiology procedures using aseptic techniques for transfer, isolation and observation of commonly encountered, clinically significant bacteria. 1
Figure 3.4 Approximate size of various types of cells. ~25 um Red Blood Cells 1.5mm 1500 um = 1500 Width of penny Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes. 3 General Principles of Microscopy Magnification Resolution Contrast Official definitions Magnification, the ratio of an object s image size to its real size Resolution, the measure of the clarity of the image, or the minimum distance of two distinguishable points Contrast, visible differences in brightness between parts of the sample 2
3 General Principles of Microscopy A) Magnification (enlarge) B) Resolution (tell apart 2 objects close together) C) Contrast (Differences in intensity between two objects) How to increase all 3 of these? A) Magnification vs B) Resolution High Magnification but Low Resolution How to increase A) Magnification and B) Resolution VIBGYOR Smaller the object = Use smaller wavelength 3
50 µm General rule for any microscopy/detector Smaller the wavelength smaller the object you can see Light vs Electron microscope Visible Light Electron has very small wavelength compared to visible light Light vs Electron microscope Visible Light Electron Magnifies objects 1000X Magnifies objects 100,000X C) Contrast (Differences in intensity between two objects) Staining increases contrast Brightfield (unstained specimen) Brightfield (stained specimen) 4
Figure 4.16 Simple stains. Light Microscopy In a light microscope (LM), visible light is passed through a specimen and then through glass lenses Glass lenses focuses light and enlarges and resolves objects Light changes direction (refracts) when it moves from one medium to another Light Air Air Glass 5
polish glass Magnification = 50/5 = 10X Focal point 5 50 Specimen Convex lens Magnify using lenses Inverted, reversed, and Enlarged image Light Microscopy Bright-field microscopes Simple Microscopes Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms for the first time in history Fresh pond water Light Microscopy (Bright-field microscopes) Compound multiple lenses Series of lenses for magnification Light passes through specimen into objective lens Have one or two ocular lenses Total magnification = magnification of objective lens X magnification of ocular lens e.g. 10 times X 10 times = 100 times 6
Figure 4.4 A bright-field, compound light microscope. occular lens Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base objective lens Figure 4.5 The effect of immersion oil on resolution. Oil immersion lens (100X) is a special type of objective lens Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip Immersion oil Slide Slide Specimen Light source Without immersion oil Light source With immersion oil Oil immersion lens Increases magnification and resolution Light changes direction (refracts) when it moves from one medium to another Light Air Air Glass 7
50 µm Figure 4.5 The effect of immersion oil on resolution. Oil immersion lens (100X) is a special type of objective lens Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip Immersion oil Slide Slide Specimen Light source Without immersion oil Light source With immersion oil Oil immersion lens Increases magnification and resolution Staining increases contrast Brightfield (unstained specimen) Brightfield (stained specimen) Light Microscopy Staining Principles of Staining Dyes used as stains are usually salts Chromophore is the colored portion of the dye + + + + + ++ + + + + - - - - - + ++ + - + + + + + - -+ + + - - - + + + Basic dyes Basic dyes (positively charged) stain acidic (negatively charged) structures Acidic dyes (negatively charged) stain alkaline structures 8
Inside of most bacterial cells is negatively charged + Basic dyes - - -+ + + - - - + + + -- - - - - Therefore Basic dyes are more commonly used to stain bacterial cells Positive Staining Inside of most bacterial cells is negatively charged Acidic dyes - - - - - - - - - - - - - - - - - -- - - Therefore Acidic dyes are more commonly used to stain background Negative Staining Simple/Differential Staining? Positive/Negative Staining? 9
Simple/Differential Staining? Positive/Negative Staining? Negative staining Rhodospirillum rubrum R. rubrum Rhodospirillum rubrum R. rubrum This large (0.7-1.0 mm), nonpathogenic gram negative spirillum exhibits polar amphitrichous flagellation and a characteristic red pigment. 10
Binomial nomenclature naming species of living things by giving each species a name composed of two parts humans belong to the genus Homo species Homo sapiens. Homo sapiens noun-genus Capitalized adjective-specific epithet Homo sapiens Homo naledi (2015) Microbe Naming Rules Each species has a unique name Genus specific epithet G. specific epithet Genus specific epithet G. specific epithet Escherichia coli Escherichia coli E. coli E. coli 11
X Microbe Naming Rules Genus specific epithet X Genus specific epithet Y Genus specific epithet Y Genus specific epithet Bacteria have shapes/morphology Figure 3.12 Bacterial shapes and arrangements. 3 major shapes 1) coccus 2) bacillus 3) spirillum 1) staphylo 2) strepto 2 major arrangements 12
cocci bacilli shape arrangement Streptococci Streptobacilli Staphylococci Microbe Naming Rules based on shape and arrangement (Arrangement+shape) (specific epithet) Streptobacillus (specific epithet) Staphylococcus (specific epithet) Light Microscopy Staining Simple stains = single stains Can be used for Positive or Negative Stains Positive Stain = any bacteria Negative Stain = any bacteria/capsule/flagella 13
Light Microscopy Staining Differential stains = multiple stains Gram stain/acid-fast stain/endospore stain Gram stain Microscopy Light Microscopy 1) Simple Staining Preparing a specimen for staining Aseptic technique Innoculating loop Inoculum (small amount of culture) 14
Prokaryotic Microbes Bacteria with 3 shapes/morphology Prokaryotic Microbes bacteria Bacteria cell model 15
Bacteria Prokaryotic Microbes vs Eukaryotic Microbes Protozoa - Paramecium Algae - Spirogyra Fungus - Yeast Eukaryotic Microbes paramecium 16
Eukaryotic Microbes spirogyra Eukaryotic Microbes yeast Culturing Microorganisms Culture Act of cultivating microorganisms or the microorganisms that are cultivated Mixed Culture vs Pure Culture Inoculum (small amount of culture) introduced into medium 17
Only live bacteria will grow and form colony Inoculum (small amount of culture) 1 bacteria = 1 COLONY Therefore bacteria also called CFU = COLONY forming unit colony composed of cells arising from a single progenitor therefore progenitor bacteria is termed a colony-forming unit (CFU) 10 COLONIES Therefore original culture had how many COLONY forming units? How many live bacteria? Mixed Culture 1 bacteria = 1 COLONY Separate bacteria from each other Mixed Culture? Pure Culture 18
2 methods to acquire pure cultures from mixed cultures Streak plates Pour plates The streak-plate method of isolation Can individually pick single cell of some large microorganisms and use to establish a culture The pour-plate method of isolation Sequential inoculations 1.0 ml 1.0 ml 1.0 ml Initial sample 9 ml broth 9 ml broth 9 ml broth 1.0 ml to each Petri dish, add 9 ml warm agar, swirl gently to mix Colonies Fewer colonies 19