BIO244 Lab Sample Outline (Your outline does not need to be typed!) Lab 2 - Goals and Objectives : Learn aseptic technique and pure culture isolation Exercise 9 and 10 Wash bench with disinfectant Exercise 9: Aseptic Technique 1. Label all tubes using tape with name, date, name of organism, and “Ex.9” 2. Perform a broth culture to broth culture aseptic transfer using E. coli: follow directions in figure ….. and ……. pages ………. Incubate at 37°C. 3. Perform a slant culture to slant culture aseptic transfer using E. coli: follow directions in figure ……. pages ………. Incubate at 30°C. 4. Perform a plate culture to slant culture aseptic transfer using S. marcescens: follow directions in figure ……. page ……….. Incubate at 30°C. 5. incubate at correct indicated temperature. 6. Remove tape from E. coli and S. marcescens cultures and discard. Exercice 10: Pure Culture Technique 1. Label plates directly with marker with name, date, “mixed culture”, and “Ex.10” and 2. Perform a streak for isolation using Quadrant Method B. Start with directions in figure …… on pages …….., then perform isolation as described on page 85: Quadrant Streak (Method B) 3. Repeat on a second plate. 4. incubate at 25°C 5. Remove tape from mixed culture and discard. 6Return all materials and wash bench with disinfectant Conclusions: 1, 2. Materials: Broth culture E. coli Slant culture E. coli Plate culture S. marcescens 1 sterile BHI broth 2 sterile BHIA slants Flame, loop, tape, marker Materials: Mixed culture (contadins E. coli, M. luteus, S. marcescens) 2 sterile BHIA plates Flame, loop, marker

Lab 1 Goals and Objectives: Learn how to use microscope and measurements Microscopy lecture: Text Chapter 3 Exercise 1: Brightfield Microscopy Exercise 5: Microscopic Measurements Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55) –Homework: “Testing Household Surface” handout, –Take home plates, swabs, and sterile water READ DIRECTIONS CAREFULLY

International measurement system Microorganisms are measured in micrometer range - µm meter- m - Standard unit of length=3.38 ft or 1.09 yd Prefix Symbol Factor Numerically Name Gram Liter Meter 1 milli m mm one thousandth micro μ µm one millionth nano n nm one billionth 1 m = 100 cm = 1000 mm = 1,000,000 µm = 1,000,000,000 nm = 10,000,000,000 Ǻ (centimeter) (millimeter) (micrometer) (nanometer) (Angstroms) cells viruses small molecules large organelles small organelles atoms

A microscope (Greek: μικρόν (micron) = small + σκοπε ῖ ν (skopein) = to look or see 1. Magnification – the ability to make larger –10X, 20X, 40X, 100X, 200X, 1000X 2. Resolution - the ability of the lenses to distinguish two points. –A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm. –Shorter wavelengths of light provide greater resolution. Microscopy: The Instruments

Microscopy Microscopes can largely be separated into three classes: 1. Light microscopes - Visible wavelengths of light - UV light - Magnification - up to 2000X - Resolution – 0.2 µm (200 nm) 2. Electron microscopes –Uses beams of electrons instead of light. –The shorter wavelength of electrons gives greater resolution. –Magnification limit – 10, ,000 X –Resolution limit – 2.5 nm ( µm) 3. Scanning probe microscopes - A physical probe is used either in close contact to the sample or nearly touching it - Resolution 1/100 of an atom.

Figure 3.2 Microscopes and Magnification. Tick Actual size Red blood cells E. coli bacteria T-even bacteriophages (viruses) DNA double helix Unaided eye ≥ 200  m Light microscope 200 nm – 10 mm Scanning electron microscope 10 nm – 1 mm Transmission electron microscope 10 pm – 100  m Atomic force microscope 0.1 nm – 10nm

Light Microscopy The use of any kind of microscope that uses light to observe specimens Types of light microscopy –Compound light microscopy –Dark field microscopy –Phase-contrast microscopy –Differential interference contrast microscopy –Fluorescence microscopy –Confocal microscopy

Figure 1.2

The image from the objective lens is magnified again by the ocular lens. Total magnification = objective lens  ocular lens Limits: Magnification – X Resolution – 0.2 µm Compound light microscope Figure 3.1b

Refractive index is the light-bending ability of a medium. The light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending. 100X objective lens 1000x total magnification Compound light microscope Figure 3.3

Brightfield Illumination Darkfield Illumination Phase-Contrast Microscopy Dark objects are visible against a bright background. –Light reflected off the specimen does not enter the objective lens. Figure 3.4a, b Accentuates diffraction of the light that passes through a specimen. Good resolution Light objects are visible against a dark background. Light reflected off the specimen enters the objective lens.

Fluorescence Microscopy Uses UV light. –Fluorescent substances absorb UV light and emit visible light. –Cells may be stained with fluorescent dyes (fluorochromes). Confocal Microscopy – Uses fluorochromes and a laser light. The laser illuminates each plane in a specimen (a slice) produce 2D or 3D image. Figure 3.6b

Ultra thin sections of specimens. Light passes through specimen, then an electromagnetic lens, to a screen or film. Specimens may be stained with heavy metal salts. 10, ,000X; resolution 2.5 nm Transmission Electron Microscopy (TEM) Figure 3.8a

Scanning Electron Microscopy (SEM) An electron gun produces a beam of electrons that scans the surface of a whole specimen. Secondary electrons emitted from the specimen produce the image ,00X; resolution 20 nm Figure 3.8b

Scanning-Probe Microscopy Scanning tunneling microscopy uses a metal probe to scan a specimen. –Resolution 1/100 of an atom. Atomic force microscopy uses a metal and diamond probe inserted into the specimen. –Produces 3D images. Figure 3.9a

Preparation of Specimens for Light Microscopy Live or unstained cells - have little contrast with the surrounding medium. –However, researchers do make discoveries about cell behavior looking at live specimens. Specimen preparation 1.Smear - A thin film of a microbes suspension on a slide 2.Dry the smear – to prevent cell lysis 3.Fix the smear - to attach the microbes to the slide

Stains consist of a positive and negative ion. In a basic dye, the chromophore is a cation. In an acidic dye, the chromophore is an anion. Staining the background instead of the cell is called negative staining. Smears staining

General categories of stain 1. Simple stain- Use of a single basic dye –methyleneblue, carbolfucsin, crystal violet –can see the size and shape of the bacteria 2. Differential Stains - use two or more stains and categorize cells into groups –Gram stain –Acid-Fast Stain

3. Special Stains –Negative staining - the background is stained, leaving the actual specimen untouched, and thus visible –Capsule staining - is useful for capsules. –Heat is required to drive a stain into endospores. –Flagella staining requires a mordant to make the flagella wide enough to see. General categories of stain Figure 3.12a-c

BIO244 Lab 1 Microscopy Students will need: microscope and cord (sign out number, use all semester) 1 “e” slide 1 stage micrometer immersion oil: share one bottle per student pair lens tissue & methanol laboratory supplemental packet lab manual or photocopied pages BIO244 Microscope Worksheet Chapter 3 Notes outline 2 sterile agar plates, stack and tape to keep lids shut 4 sterile swabs 1 vial sterile water

Exercise 1: Brightfield Microscopy –Learn how to use the microscope (guide in suppl. packet p.51) 1. Diopter and interocular adjustments 2. Light adjustments (iris diaphragm) 3. Oil immersion

Exercise 5: Microscopic Measurements –Calibrate the ocular micrometer (suppl. packet p.52) –Microscope Worksheet –Measure the provided specimens –Complete the microscope worksheet and turn it in –Microscope Measurement Concept Practice Homework: complete –Measurement practice sheet in supplemental packet, self check: answers in packet (p )

Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (suppl. packet p.52)

1.Make sure left ocular is turned fully counter-clockwise 2.Start with 10X objective 3.Get stage micrometer (2mm ruler on slide) in focus: -you will see the ocular micrometer with labels in multiples of 10 (0, 10, 20, 30) -the stage micrometer with labels in multiples of 0.1 (0.0, 0.1, 0.2) stage micrometer ocular micrometer

3. Line up the zero lines of the two micrometers (you can turn the top of the ocular to position the ocular micrometer and the mechanical stage controls to move the stage micrometer) At 100X total magnification they should line up as shown below “ 0.1” on stage micrometer is 0.1mm on that actual 2mm ruler. This distance corresponds to 10 “units” (spaces between lines) on the ocular micrometer (0-10): the 0.1 line of the stage ruler lines up with the 10 line on the ocular one. How long in millimeters is one unit on the ocular micrometer with the 10X objective in place?

0.1mm = 10 “units” 1 unit = 0.1mm ÷ 10 1 unit = 0.01mm to convert mm to µm, multiply by 1000 When the microscope is set to 100X total magnification, one unit on the ocular micrometer is worth ________µm

4. Repeat for the 40X objective :

0.1mm = 40 “units” 1 unit = 0.1mm ÷ 40 1 unit = mm to convert mm to µm, multiply by 1000 When the microscope is set to 400X total magnification, one unit on the ocular micrometer is worth ________µm

When the microscope is set to 100X total magnification, one unit on the ocular micrometer is worth ____10____µm When the microscope is set to 400X total magnification, one unit on the ocular micrometer is worth ____2.5____µm When the microscope is set to 1000X total magnification, one unit on the ocular micrometer is worth ____1____µm

Dinoflagellate 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

Lilium Pollen 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

Frog Blood 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

100X objective (1000X) 40X objective (400X) Streptococcus faecalis Czura 2005

Dinoflagellate Frog Blood Lilium Pollen Streptococcus faecalis

Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55) –Homework: “Testing Household Surface” handout, –Take home plates, swabs, and sterile water READ DIRECTIONS CAREFULLY