MCB 100 Exam 1 Spring 2019 Wednesday February

MCB 100 Exam 1 Spring 2019 Wednesday, February 13, 2019 2: 00 – 2: 50 pm Covers: chapters 1, 2, 4, 3 & 6 (Chapter 5 is not on exam 1!) Place: If your last name take the exam in starts with A – Pe: please room 112 Gregory starts with Ph – Z: please room 213 Gregory Review Session: 7: 00 – 8: 20 pm, Monday, Feb. 11 Place: Room 124 Burrill Hall

MCB 100 Exam 1 Spring 2019 Practice Exam 1 has been updated. To find the link to the practice exam, go to: www. life. illinois. edu/mcb/100 - Click on: “Exam Information” - Scroll down to find the link to the practice exam. (It downloads as a MS Word Document. )

Do you have a conflict for Exam 1, which is scheduled for 2 – 2: 50 pm on Wed. 2/13/2019? Contact Dr. Chapman by e-mail: kenchap. life. illinois. edu What is the nature of your conflict? The conflict exam will be given at 3 pm or 5 pm on Tuesday, 2/12/19 in room 242 Burrill Hall. Does that fit into your schedule? (It's possible to do it earlier in the day but it's more difficult to find a quiet room where a student can take an exam when the lab is being used by a class. ) Conflict exams must be taken before the rest of the class takes the test. Sick on the day of the exam? Don’t come and fail the test because you feel so bad you can’t think. See a doctor and take care of your health. Also get an excuse note. There are no make-up exams in MCB 100, but in the event of illness or unexpected circumstances your score can be prorated. If you take a test, your score will be counted as it is. You can’t take a test and later decide you’d rather have your score prorated.

MCB 100 Introductory Microbiology Chapter 4 Microscopy Wednesday, February 6, 2019

Anthony Van Leeuwenhoek used a home-made simple microscope to make the first reported observations of bacteria and other microbes in the late 1600 s. Late 1600 s Robert Hooke used compound microscopes to examine small plant and animal specimens. He observed the compartmentalized structure of cork and introduced the use of the term “cell” in biology.

Magnification: the ability of a microscope or a lens to increase the apparent size of the image of an object. Total Magnification: For a compound microscope the total magnification is the product of the magnifying abilities of all of the lenses in the system. Magnification Total Magnification = of the Objective Lens x Magnification of the Ocular Lens

Total Magnification of a Compound Microscope If the magnifying power of the objective lens is 90 x and the magnifying power of the ocular lens is 11 x, what is the total magnification? A. 79 x B. 101 x D. 1199 x C. 990 x E. 9011 x

Resolution: The ability to discern fine details in an image of an object. Resolving Power: (AKA: Resolution Distance) R. P. is a measure of the smallest distance between two separate objects that can be seen to be two separate objects. The Resolving Power Formula R. P. = 0. 61 x wavelength___ numerical aperture Smaller R. P. = ability to see finer details To get better resolution (a smaller R. P. ) you need to increase the numerical aperture or decrease the wavelength of the light used to illuminate the object.

The Limits of Resolution with Light Microscopy Typical bacterial cells are 700 – 3000 nm in length. Typical viruses are smaller than 100 nm in diameter. For light with a wavelength of 480 nm (blue) using a good microscope with a numerical aperature of 1. 25… Resolving Power = 0. 61 x 480 nm / 1. 25 = 235 nm - Viruses are too small to see with a light microscope and bacteria are so small you can’t see much detail.

Microscopy Techniques A. Bright-field Microscopy B. Dark-field Microscopy C. Phase Contrast Microscopy D. Fluorescence Microscopy E. Electron Microscopy

Bright-field Microscopy Light shines through the specimen and directly into the objective lens and to the observer’s eye creating a lighted or bright field of vision. Bacteria and other microorganisms are seen as dark objects on a white background. This works best when the bacteria are stained a bright color and immobilized. Bright-field microscopy has the advantage of being easy to use. The disadvantage is that it works best with fixed and stained microorganisms (dead). Dark-field Microscopy Light shines on the specimen from an oblique angle and does not go directly into the objective lens. The background or field of vision is dark. The only light that gets to the observer’s eye is the light that is scattered by the specimen. The specimen does not have to be stained. This technique works for live microorganisms but they move around pretty fast making it difficult to focus on them.

Microscopy 1 Choose the FALSE statement about microscopy. A. One can’t see viruses with a light microscope even if the objective lens is of excellent quality. B. You can get better resolution with a light microscope by using red light rather than blue light. C. If you want to see unstained bacteria dark field microscopy is better than bright field microscopy. D. The quality of a compound microscope is determined by the quality of the worst lens in the system. E. The Gram stain was invented in 1884.

Microscopy 1 Choose the FALSE statement about microscopy. A. One can’t see viruses with a light microscope even if the objective lens is of excellent quality. B. You can get better resolution with a light microscope by using red light rather than blue light. FALSE C. If you want to see unstained bacteria dark field microscopy is better than bright field microscopy. D. The quality of a compound microscope is determined by the quality of the worst lens in the system. E. The Gram stain was invented in 1884.

Phase Contrast Microscopy Invented in 1934 by Frits Zernike, phase contrast microscopy relies on a complicated optical trick to enhance the visual effect seen when light goes from a medium of lesser optical density (water) to a medium of greater optical density (cytoplasm). Beams of light are bent when they cross the boundary between two different transparent substances with different optical densities. Phase Contrast Microscopy is most useful for examining living protozoa and other eukaryotic cells. It does not require the specimen to be stained. Epithelial cell in bright field microscopy vs. phase contrast. Source: wikipedia

Fluorescent Microscopy In Fluorescent Microscopy the specimen is illuminated with UV light which can cause some chemicals to emit visible light. UV light is called black light because you can’t see it. The only visible light seen is that which is produced by fluorescence of naturally occurring compounds in the microorganism or special dyes that are used to stain the specimen.

Natural fluorescence - Some bacteria (ex: Pseudomonas aeruginosa) make a fluorescent pigment - Photosynthetic pigments like chlorophyll are fluorescent - Methanogenic archaea make a fluorescent pigment Fluorescent dyes for DNA (acridine orange) for Bacillus anthracis (fluorescein isothiocyanate) for Mycobacterium tuberculosis (auramine O) Immunofluorescence microscopy A fluorescent dye such as fluorescein (yellow-green) or rhodamine (red) is chemically joined to an antibody to make a very specific probe. The green algae Chlamydomonas stained with 2 fluorescent antibodies. Merchant et al. in Science 10/12/2007

Microscopic Techniques What type of microscopy would be the most likely to be used by a pathologist who wants to examine a tissue sample for the presence of abnormal-looking (cancerous) cells? A. B. C. D. Dark Field Microscopy Bright Field Microscopy Phase Contrast Microscopy Immunofluorescence Microscopy

Electron Microscopy - General features: The sample is bombarded with electrons The image is produced using a cathode ray tube Sample must be dehydrated Sample must be stained with a heavy metal Sample must be in an evacuated chamber TEM - Transmission Electron Microscopy - thin sliced sample - used to see cellular organelles such as E. R. , mitochondria, chloroplasts, ribosomes etc. SEM - Scanning Electron Microscopy - sample is sputter coated with gold used to look at the surface of objects (variant = freeze-fracture etching)

Stains for Light Microscopy Simple Stains Basic dyes are used to stain the microorganisms. The chromaphore has a positive charge & binds to negatively charged groups on the bacterial cell wall. Negative Stains Acidic dyes are used to stain the area around the microorganism. The chromaphore has a negative charge and sticks to glass. Differential Stains Two or more dyes are used and different microorganisms stain different colors. This is caused by differences in the structure or chemical make-up of the cell surface of the microorganisms. Examples of Differential Stains - The Gram Stain - Schaeffer-Fulton Endospore Stain - Ziehl-Neelsen Acid-Fast Stain

The Gram Stain The Gram stain is used to divide bacteria into two groups based on the thickness of the cell wall. Bacteria with thick cell walls will appear purple and are called Gram positive. Bacteria with thin cell walls will appear pink and are called Gram negative. Examples of Gram Positive Bacteria: Clostridium tetani, Streptococcus pneumonia, Corynebacterium diphtheria Examples of Gram Negative Bacteria: Esherichia coli, Salmonella sp. Vibrio cholera, Yersinia Pestis

Ziehl-Neelsen Acid-Fast Stain The Acid-Fast stain is used to identify bacteria in the Genera Mycobacteria and Nocardia, including the clinically very significant bacterium Mycobacterium tuberculosis. The basis of this differential stain is that the cell walls of the Mycobacteria contain a lot of hydrophobic waxy mycolic acids. The dye Carbol Fuchsin sticks tightly to the waxy mycolic acids. Decolorization with acidified alcohol removes the Carbol Fuchsin from other types of bacteria. The counterstain is typically methylene blue.

Schaeffer-Fulton Endospore Stain The Endospore Stain is used to identify spore forming bacteria in the Gram positive genera Bacillus and Clostridium. It is especially useful for identifying: Clostridium botulinum, Clostridium tetani, C. perfringens & Bacillus anthracis. The endospore cell wall is mineralized with dipicolinic acid and calcium. The endospore can be stained by the dye Malachite Green. Most bacterial cells don’t stain well with Malachite Green. The red dye Safranin is used as a counterstain to see the vegetative (non-spore) cells. Endospore Stain Vegetative cells (actively growing) stain red. Spores are green.

Stains for Microscopy What is the purpose of the Ziehl-Neelson Acid Fast stain? A. helps to identify Clostridium and Bacillus species B. helps one to detect the presence of a capsule around bacterial cells C. separates bacteria into two groups based on thickness of the cell wall D. helps to identify Mycobacterium species like tuberculosis bacteria