Types of mounting of specimens to a slide

1. Types of mounting of specimens to a slide • Wet mount – specimen suspended in drop of water • Mix water with alkali to dissolve background material – KOH method • Add alkali and dye – lactophenol cotton blue, iodine • Dyes nonspecifically stain cellular material, increasing contrast with background • India ink method – darkens background rather than cell. Capsule stain

2. Differential stains • Gram stain – best known and most widely used. Provides phenotypic classification. • Iron hematoxylin and trichrome stains invaluable for identifying protozoan parasites. • Wright-Giesma stain – identify blood parasites

3. Acid-Fast stains • Some organisms retain a primary stain even when exposed to a strong decolorizing agent such as an acid or alcohol. • Ziehl-Neelsen – oldest method. Requires heating the specimen during the staining procedure. • Kinyoun method – Cold acid-fast stain. No heating requirement. • Auramine-rhodamine method – stain of choice. Large area of specimen can be examined rapidly by searching for fluorescing organisms against a black background.

4. Fluorescent stains • Acridine orange stain – stain bacteria and fungi • Calcofluor white – stains chitin in fungal cell walls • Fluorescent antibody stains – specific antibodies attached to specimen labeled wit fluorescent dyes. Sensitive and specific.

5. Characteristics generally present in a Gram-positive bacterium • cytoplasmic lipid membrane • thick peptidoglycan layer • Teichoic acids and lipoids are present, forming lipoteichoic acids which serve to act as chelating agents, and also for certain types of adherence. • Most pathogenic bacteria in humans are Gram-positive organisms.

7. Medical Microbiology What it’s about? The little critters that make us sick and those that keep us healthy. How they tick. What makes them work? Why are they important to know about?

8. Textbooks are available to check out as long as you bring them back the next day as they may be part of the class work that day.

9. Nosocomial Infections • A Nosocomial Infection is a hospital- or clinic-acquired Infectious Disease. • Nobody goes into medicine in order to prevent Nosocomial Infections. • Nevertheless, to practice medicine you must be able to prevent Nosocomial Infections. • To prevent Nosomial Infections you need to have some understanding of microbiology. • Oh yes, and Infectious Disease, in general,is kind of important to medicine, too. • At the very least, you should be striving to Do No Harm! • And avoiding harming by infection requires some reasonable knowledge of microbiology.

10. What is Microbiology? • Microbiology is the Science that studies Microorganisms. • Microorganisms, roughly, are those living things that are too small to be seen with the naked eye. • Microorganisms cannot be distinguished Phylogenetically from “Macroorganisms” • For example, many fungi are microorganisms, as well as all bacteria, all viruses, and most protists. • Microbiology is more a collection of techniques: • Aseptic technique • Pure culture technique • Microscopic observation of whole organisms • A microbiologist usually first isolates a specific microorganism from a population and then cultures it (i.e., in pure culture).

11. Origin of Microbes: Spontaneous Generation Myths • Snakes from horse hairs in stagnant water • Mice from grain and cheese wrapped in a sweater • Maggots from rotting meat • Fleas from hair • Flies from fresh and rotting fruit • Mosquitoes from stagnant pondwater • Eels from slimy mud at the bottom of the ocean • Locusts from green leaves • Raccoons from hollow tree trunks • Termites are generated from rotting wood

12. Pasteur was fortunate to have worked with broths prepared from non-soil or -plant associated substances (e.g., hay). • Those substances containbacteria that can form endospores, not all bacteria can. • Endospores represent a bacterial durable state and are very difficult to kill. • John Tyndal (1876) discovered that there exist differences in the ability of heat to kill different kinds of bacteria-containing cultures. • Ferdinand Cohn(1876) showed that this difference was due to endospores andRobert Koch (1877) showed that the bacterium Bacillus anthracis forms endosporesas part of its transmission. Problem of Endospores

13. Types of Microorganisms • Bacteria • a.k.a., eubacteria (“true” bacteria) • a.k.a., domain Bacteria • Archaeabacteria • a.k.a., domain Archaea • Single-celled members of domain Eukarya. • Protozoa • Microscopic Algae • Microscopic Fungi • Viruses and other Agents

14. Relationship of Microbes

15. Comparing Domains

16. Types: Bacteria Description:eubacteria, archaeabacteria, Gram-negative, Gram-positive, acid fast,cyanobacteria Types: prokaryotes, absorbers, wet conditions, animal decomposers, cell walls, unicellular Nutrient Type:chemoheterotrophs, photoheterotrophs, chemoautotrophs, photoautotrophs Durable state: endospores (some) Diseases:tetanus, botulism, gonorrhea, chlamydia, tuberculosis, etc., etc., etc.

17. Binomial Nomenclature (1/3) Examples: Escherichia coli, E. coli, Escherichia spp., and “the genus Escherichia” The genus name (Escherichia) is always capitalized The species name (coli) is never capitalized The species name is never used without the genus name (e.g., coli standing alone, by itself, is a mistake!) The genus name may be used without the species name(e.g., Escherichia may stand alone, though when doing so it no longer actually describes a species) When both genus and species names are present, the genus name always comes first(e.g., Escherichia coli, not coli Escherichia)

18. Binomial Nomenclature (2/3) Both the genus and species names are always italicized (or underlined)—always underline if writing binomials by hand The first time a binomial is used in a work, it must be spelled out in its entirety (e.g., E. coli standing alone in a manuscript is not acceptable unless you have already written Escherichia coli in the manuscript) The next time a biniomial is used it may be abbreviated (e.g., E. for Escherichia) though this is done typically only when used in combination with the species name (e.g., E. coli) The species name (e.g., coli) is never abbreviated

19. Binomial Nomenclature (3/3) It is a good idea to abbreviate unambiguously if there is any potential for confusion (e.g., Enterococcus vs. Escherichia) These rules are to be followed when employing binomial nomenclature even in your speech. It is proper to refer to Escherichia coli as E. coli or even as Escherichia, but it is not proper to call it coli or E.C.! When in doubt, write the whole thing out (and underline)!

20. Types: Cyanobacteria Description: also called blue-green algae, cyanobacteria are a kind of bacteria (more specifically, a kind of eubacteria) Types: photosynthetic aquatic procaryotes,green lake scum,cell walls Nutrient Type: photoautotrophs Durable state: ? Diseases: none

21. Types: Algae Description: photosynthetic aquatic eukaryotes, cell walls, both unicellular and multicellular types Types: brown, red, green, diatoms, dinoflagellates, euglenoids Nutrient Type: photoautotrophs Durable state:? Diseases: Some poisonings associated with unicellular types: Alexandrium causes Paralytic Shellfish Poisoning (PSP), Dinophysis causes Diarrhetic Shellfish Poisoning (DSP), Pseudo-nitzschiamultiseries causes Amnesic Shellfish Poisoning (ASP) [some would describe some as protists]

22. Types: Fungi Description: yeasts (unicellular fungi), molds (filamentous fungi) Types: eucaryotes, nutrient absorbers, dry conditions, plant decomposers, cell walls,~100 human pathogens Nutrient Type: chemoheterotrophs Durable state: spores (not endospores) Diseases: mycoses: candida, ringworm (pictured), athlete's foot, jock itch, etc.

23. Types: Helminths Description: Flatworms(platyhelminths),roundworms(nematodes) Types: metazoan (multicellular animal) parasites, engulfers and absorbers Nutrient Type: chemoheterotrophs Durable state:? Diseases: trichinosis, hook worm, tape worm (pictured are scolex-heads of), etc.

24. Types: Protozoa (Protists) Description: Unicellularand slime molds, flagellates, ciliates Types: eucaryotes, parasites (most not),engulfers and absorbers, wet conditions, no cell wall, ~30 human pathogens Nutrient Type: chemoheterotrophs (some classifications include some photoautotrophs as well) Durable state: cysts (some) Diseases: malaria, giardiasis, amoebic dysentery, etc. (shown are harmless--to us--protist components of pond water: Amoeba, Blepharisma, Paramecium, Peranema, & Stentor)

25. Types: Viruses Description: Viruses are not cells but some viruses do have lipid envelopes Types: acellular, obligate intracellular parasites Nutrient Type: not applicable Durable state: virion particles, some can encase in durable state of host Diseases: common cold, flu, HIV, herpes, chicken pox, etc.

26. Other “Agents”

27. Microbes & Ecology • Microbes are produces—they provide energy to ecosystems, especially aquatic ecosystems • Microbes are fixers—they make nutrients available from inorganic sources, e.g., nitrogen • Microbes are decomposers—they free up nutrients from no longer living sources • Microbes form symbioses(such as mycorrhizal fungi associated with plant roots—though somewhat macroscopic, the bacteria found in legume root nodules, etc.) • Microbes serve as emdosymbionts (e.g., chloroplasts and mitochondria)

28. Microbes & Industry • Industry: Fermentation products (ethanol, acetone, etc.) • Food: Wine, cheese, yogurt, bread, half-sour pickles, etc. • Biotech: Recombinant products (e.g., human insulin, vaccines) • Environment: Bioremediation Each carton of Bugs+Plus provides easy to follow step-by-step instructions, containers of specially-formulated wet and dry nutrients and a container of microbes cultured for their ability to digest oil and other petroleum derivatives.

29. Microbes & Disease • Microbes both cause and prevent diseases • Microbes produce antibiotics used to treat diseases • The single most important achievement of modern medicine is the ability to treat or prevent microbial disease • Most of this course will consider the physiology of microbes and their role in disease • The Germ Theory of Disease = Microbes cause disease! • (yes, it wasn’t so long ago that humans didn’t know this) • Nevertheless, most microorganisms, including most bacteria, do not cause diseases in any organism (including in humans)

30. Impact of Infectious Disease • Infectious diseases are diseases caused by microbes • 200,000 deaths per year in U.S. from infectious diseases • ~20 million died from influenza (a disease caused by a virus) in 1918 • “New” infectious diseases still being discovered

31. Normal Flora These are the ~harmless microorganisms found on your body. Every part of your body that normally comes in contact with outside world (deep lungs and stomach are exceptions).