1. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Burton's Microbiology for the Health Sciences Chapter 2. Viewing the Microbial World

2. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 2 Outline Introduction Using the metric system to express the sizes of microbes Microscopes –Simple microscopes –Compound microscopes –Electron microscopes –Atomic force microscopes

3. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Using the Metric System to Express the Sizes of Microorganisms Metric units are used to express the sizes of microbes. The basic unit of length in the metric system is the meter (m); it is equivalent to 39.4 inches. The sizes of bacteria and protozoa are usually expressed in terms of micrometers (µm). A micrometer is one millionth of a meter. A typical spherical bacterium (coccus) is approximately 1 µm in diameter. A typical rod-shaped bacterium (bacillus) is approximately 1 µm wide by 3 µm long.

4. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Representations of Metric Units of Measure and Numbers

5. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Using the Metric System to Express the Sizes of Microorganisms, cont. The sizes of viruses are expressed in terms of nanometers (nm). A nanometer is equal to one billionth of a meter. Most of the viruses that cause human diseases range in size from 10 nm to 300 nm. One exception is Ebola virus, a cause of viral hemorrhagic fever. Ebola viruses can be as long as 1,000 nm (1 µm). When using a microscope, the sizes of microorganisms are measured using an ocular micrometer.

6. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Microscopes The human eye, a telescope, a pair of binoculars, a magnifying glass and a microscope are various types of optical instruments. A microscope is an optical instrument that is used to observe tiny objects; objects so small that they cannot be seen with the unaided human eye. Each optical instrument has a limit as to what can be seen using that instrument; this limit is referred to as the resolving power or resolution of the instrument. The resolving power of the unaided human eye is approximately 0.2 mm.

7. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Early Microscopes

8. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Simple Microscopes A simple microscope is one that contains only one magnifying lens. A magnifying glass could be considered a simple microscope; when using a magnifying glass, images appear 3-20 times larger than the object’s actual size. Leeuwenhoek’s simple microscopes had a maximum magnifying power of about X300 (about 300 times).

9. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Compound Microscopes A compound microscope contains more than one magnifying lens. Because visible light is the source of illumination, a compound microscope is also referred to as a compound light microscope. Compound light microscopes usually magnify objects about 1000 times. The resolving power of a compound light microscope is approximate 0.2 µm (about 1,000 times better than the resolving power of the unaided human eye).

10. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Compound Microscopes, cont. It is the wavelength of visible light (~0.45 µm) that limits the size of objects that can be seen. Objects cannot be seen if they are smaller than half of the wavelength of visible light. Today’s laboratory microscope contains two magnifying lens systems: –The eyepiece or ocular lens (usually X10) –The objective lens (X4, X10, X40, and X100 are the four most commonly used objective lenses)

11. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins A Modern Compound Microscope

12. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Compound Microscopes, cont. Total magnification is calculated by multiplying the magnifying power of the ocular lens by the magnifying power of the objective lens being used. –X10 ocular x X4 objective = X40 total mag. –X10 ocular x X10 objective = X100 total mag. –X10 ocular x X40 objective = X400 total mag. –X10 ocular x X100 objective = X1000 total mag. Photographs taken through the lens system of the compound light microscope are called photomicrographs.

13. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Compound Microscopes, cont. Because objects are observed against a bright background or “bright field,” the compound light microscope is sometimes referred to as a brightfield microscope. If the condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background or “dark field;” the microscope is now called a darkfield microscope. Other types of compound microscopes include: –Phase contrast microscopes –Fluorescence microscopes

14. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Darkfield Microscopy of Treponema pallidum (the bacterium that causes syphilis)

15. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Phase Contrast and Fluorescent Microscopes Phase contrast microscopes are used to observe unstained living microorganisms. –Organisms are more easily seen because the light refracted by living cells is different from the light refracted by the surrounding medium. Fluorescent microscope contains a built-in ultraviolet (UV) light source. –When UV light strikes certain dyes and pigments these substances emit a longer wavelength light causing them to glow against a dark background.

16. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Electron Microscopes Electron microscopes enable us to see extremely small microbes such as rabies and smallpox viruses. Living organisms cannot be observed using an electron microscope – the processing procedures kill the organisms. An electron beam is used as the source of illumination and magnets are used to focus the beam. Electron microscopes have a much higher resolving power than compound light microscopes. There are 2 types of electron microscopes - transmission and scanning.

17. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Transmission Electron Microscope Uses an electron gun to fire a beam of electrons through an extremely thin specimen (<1 µm thick). An image of the specimen is produced on a phosphor- coated screen. Magnification is approx. 1000 times greater than the compound light microscope. Resolving power is approx. 0.2 nm.

18. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Scanning Electron Microscope

19. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins S. aureus and red blood cells as seen by light microscopy S. aureus

20. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins S. aureus in the process of binary fission, as seen by transmission electron microscopy (TEM)

21. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Scanning electron micrograph of S. aureus

22. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Scanning electron microscopy reveals the corkscrew shape of the spirochete, Treponema pallidum; note the illusion of depth. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

23. Atomic Force Microscopes Enable scientists to observe living cells at extremely high magnification and resolution under physiological conditions. Can observe single live cells in aqueous solutions. Provides a true three- dimensional surface profile.