MICRO 440: Lab Exam 1 (Weeks 1-4)

What’s the proper name of pipette A?

Transfer pipette

What is the volume of a major gradation of syringe B?

1 mL

What is the volume of a minor gradation of syringe B?

0\.2

If you dispensed the fluid in syringe B, how much fluid would you have dispensed?

4 mL

To what domain does the organism in image A belong?

Domain Eukarya

With what disease or process is the organism in picture A associated?

photosynthesis

What is the proper name (Genus + specific epithet)?

*Taenia solium*

How do humans contract this organism?

Eating undercooked infected pork

What’s the primary stain of the Gram stain?

Crystal violet

If you forgot the Gram’s iodine when staining cells, what would you see and why?

Everything will be pink/red if there’s no CV-iodine precipitates. The CV will get washed from cells by decolorizing.

What are the proper technical terms to describe the morphology and arrangement of cells in the image?

Cocci in tetrads and sarcinae

What Gram staining property do the cells in the image have?

Gram positive

In the San Joaquin Valley Area, patients have developed a wracking cough following a dust storm. Examination of fluid from lungs of patients shows the presence of spherules and fibrocaseous nodules.

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**Proper name** (Genus + specific epithet) of the organism responsible for patients’ illness?

*Coccidiodies immitis*

To what **major group** (eg. Zygomycota) does this organism belong?

Ascomycota

What’s the proper name of the **fleshy fruiting body** produced by some members of the Ascomycota group?

Ascocarp

Bacteria examples?

*Bacillus anthracis; Neisseria gonorrhoeae; Nostoc; Rhizobium* (in root nodules)

Fungi ==Ascomycetes== (Ascomycota) examples?

1. *Saccharomyces cerevisiae* budding and sporulating
2. *Claviceps purpurea* - perithecial head, ergot images
3. *Candida albicans*
4. *Coccidioides imitis*
5. *Ophiocordyceps unilateralis*
6. *Sporothrix spp.*
7. *Trichophyton mentagrophytes*
8. morels

Fungi Basidiomycetes (Basidiomycota) examples?

1. button mushroom
2. *Filobasidiella neoformans (Cryptococcus neoformans)*
3. *Ustilago* maydis (huitlacoche)

Fungi ^^Zygomycetes^^ (Zygomycota) examples?

*Rhizopus* sporangia/sporangiophores and zygosporangia

Fungi examples?

Ectomycorrhizae, lichens, onychomycosis (nail fungus image), sporotrichosis; coccidioidomycosis;
ergot/ergotism

Protists examples?

1. **Plant-like (algae):** *Spirogyra*
2. **Fungus-like (water & slime molds):** *Physarum* plate; *Phytophthora* infestans
3. **Amoebozoa (amoebas):** *Amoeba proteus, Entamoeba histolytica, Amoeba* model
4. **Ciliophora (“ciliates”):** *Paramecium* model, *Paramecium, Balantidium* coli
5. **Mastigophora (“flagellates”):** *Euglena* model, *Euglena, Trypanosoma brucei, Trypanosoma cruzi, Giardia lamblia; Trichomonas vaginalis; Trichonympha*
6. **Apicomplexa (“sporozoa”):** *Plasmodium gametocyte & merozite*s (lab manual); *Toxoplasma gondii*

Animalia examples?

*Taenia solium* cysticercus larva, *Aedes*

Acellular examples?

prions (image), prion disease brain sample (image), influenza virus (image)

Spill management

1. Announce spill & stay in place; colleagues will help
2. Treat spill & dispose of material properly (glass in sharps container; towels & gloves in biobin)
3. Eyewash station: help colleagues
4. Shower in autoclave room

Autoclaving conditions (kills all cellular organisms, viruses)

121\*C, 15 psi, 15-20 mins

What can cause a host to be compromised?

immunosuppression or breaches in the skin

What are portals of entry?

eyes, nose, mouth, and mucous membranes

What important safety precautions should be taken when incubating screw-cap tubes? When placing tubes in the “kill area”?

1. incubation: tube caps loosened
2. tubes in “kill area”: caps loosened ¼ turn, tape labels removed

What info should be placed on labels? Where should labels be placed on tubes and plates?

1. name, date, and microbe.
2. Labels placed at the top of tubes
3. Plates inverted with the agar side up; label on the agar side.

How should spent cultures be prepared for disposal? Where should they be placed for disposal?

1. Plates taped shut with masking tape.
2. Tubes: tape removed, caps loosened. Autoclaving conditions are **121*C, 15 psi, and 15-20 mins** = kill all cellular organisms and viruses. Placed in the **Kill Area.**

What are opportunistic pathogens?

microbes in the soil, water, and on/in in the body that take advantage of opportunities (a compromised host) = result in disease

Amplification & infectious dose

Many microbes grow from a few, leading to infectious dose = sufficient microbes can cause a disease

Compromised hosts/events

1. Healthy pregnant woman
2. Healthy student with a cut on their hand
3. HIV-positive person
4. Person undergoing cancer chemotherapy

Inoculating loop function

Collecting and transferring small amount of culture, especially liquid culture; streaking plates to perform “dilution over distance”

Inoculating needle function

For solid cultures, moving very small amounts, stab inoculation

Bacticinerator

Gasless, flameless sterilizer used with inoculating loops and needles

Syringes

Measuring volume to transfer larger amounts

Serological pipette measures?

Major (5 mL) & minor (1 mL) gradations

Incubators

Controls metabolism and growth through temperature to culture microbes

Tubes

Culture and handle microbes, store samples; either agar slants, agar deep, broth, or plates

Plates

Grow bacteria/etc. on a medium

What is sterile or aseptic technique? What is the purpose of practicing sterile/aseptic technique?

1. When any microorganisms are removed by inducing heat; using Bacti-Cinerator to sterilize an inoculating needle or loop
2. Avoid contamination; keep pure cultures pure.

Media

plural for medium; microbe food

Broths

liquid (“soup”)

Solid

broth + gelling agent (agar)

What is this specimen and the green dots?

*Spirogyra* (protist), chloroplasts

What is this specimen?

*Elodea* (plant)

Photosynthesis equation

6 CO2 + 6 H2O → C6H12O6 + 6 O2

Define resolution & its advantage?

clarity of object’s image; ability to distinguish between close objects

Define par centrality & its advantage?

1. Lenses centered relative to each other; objects in the center of the field using one lens will be centered in the other lens. (Center the object you’re observing before switching lenses)

Define parfocality and its advantage?

Lenses %%focused%% relative to each other; only small adjustments in focus are needed when changing lenses (objectives)

Rheostat function

adjust light intensity

Condenser function

When opened & raised, it increases light focused on specimen thus increasing resolution

Fine focus function

Small-scale movements with 40x (high dry) or 100x (oil immersion) objective lenses

Condenser function

Focuses all of the light rays on specimen to maximize illumination

How to increase & adjust resolution?

1. Increase numerical aperture (measure of a lens’s ability to gather light).
2. Use **rheostat to increase light intensity,** raise the substage condenser, and open the iris diaphragm.

Contrast adjustment involves:

Increase light intensity with rheostat, lower the substage condenser, and close the iris diaphragm.

Describe the proper way to examine a bacterial smear, beginning with the scanning lens and working up to the oil immersion lens.

1. place the slide on the stage in the mechanical slide holder, center the specimen over the opening in the stage and make any distance adjustments between the two oculars to account for one’s own interpupillary distance.
2. 4x scanning lens: adjustments to the iris diaphragm for optimum illumination, contrast, and image
3. coarse-focus adjustment knob to focus the image and use fine-focus so it is in the sharpest focus, then work through the low (10x) and high-dry (40x) objectives.
4. Ensure specimen is desirably positioned before moving to the next objective, so it’s not lost at higher magnification
5. focused under high dry = rotate the nosepiece to a midway position between the high-dry and oil-immersion lenses, drop immersion oil on the specimen, and ensure oil doesn’t get on the microscope or lenses.
6. Rotate oil lens so its tip is submerged in the oil drop, pass through it, and return the oil lens into the oil to minimize air bubbles.

With which objective lenses may the coarse focus adjustment knob be used? With which objective lenses may the ^^fine focus adjustment knob^^ be used?

1. 4x and 10x lenses
2. ^^40x and 100x lenses^^

How to make a wet mount?

1. drop of water placed on the slide and organisms are introduced to it, or if the organism is already in a liquid medium then a drop of medium is placed on the slide
2. cover glass is placed over the preparation to flatten the drop and keep the objective lens from getting wet.

What are the advantages of a %%wet mount%% over a ==heat fixed and stained smear?==

1. %%quick prep;%% can flatten specimen to make it easier to see its size and shape, characteristic arrangement/grouping of cells in natural color, motility
2. ==**stained and heat-fixed smears**== can distort size, shape, and arrangement of cells making it **difficult** to identify motility b/c organism is dead.

Prion proteins; What forms are A & B in?

normal (alpha), pathogenic (beta)

What is this image depicting?

Normal brain tissue and prion-affected tissue

What virus is this?

Influenza

What is this bacteria?

*Bacillus anthracis,* endospore former

What is this bacteria?

*Neisseria gonorrhoeae* in pus (cocci in neutrophils)

*Nostoc*: What is the name of the structure at the tip of the pointer/arrow, and which unique chemical reaction occurs within such structures?

 

heterocyte; N2 -> NH3

What is the name of this protist?

*Amoeba proteus*

What’s this asexual specimen?

*Saccharomyces cerevisiae* (buds at ends of arrows)

What is this specimen?

*Spirogyra* (vegetative at far left; rest are conjugating)

What’s this specimen?

*Rhizopus* sporangia

What subcellular structure carries out photosynthesis in plants?

Chloroplasts

Function of heterocyte in Nostoc?

1. Colorless specialized cells in cyanobacteria that provide the anaerobic (oxygen-free) environment necessary for the operation of the **nitrogen-fixing** enzymes.
2. Cyanobacteria’s properties allow them to thrive in various habitats like marine and freshwater environments, soil, and rocks in different temperatures, live as unicellular organisms or in colonies, and can be filamentous (form sheaths or biofilms).

Taxonomy is?

classification, description, identification, and naming of living organisms

Phylogeny is?

grouping organisms to reflect derivation from a common ancestor; reflect evolutionary history/relations of a group of organisms

Eukaryote: Kingdom Fungi (yeasts, molds, mushrooms) examples

1. ***Saccharomyces cerevisiae:*** budding (cells at ends of arrows) = asexual reproduction; Genus name: sugar fungus; Cerevisiae: beer maker
2. ***Rhizopus sporangia:*** sporangia (look like candy part of a lollipop) = asexual reproduction structures. Brown material are spores, cause bread mold.

Eukaryote: Kingdom Animalia (multicellular, no cell wall, motile, nutrition – ingestion) examples

1. ***Taenia solium cysticerus:*** pork tapeworm, photomicrograph of a cysticerus larva; dark pink part (infectious for humans)
2. ***Aedes:*** mosquitos can be vectors for pathogens like malaria or Zika virus.
3. ***Helminths:*** multicellular parasitic worms; diseases caused involve microscopic eggs and larvae (guinea worm/Dracunculus medinensis: dizziness, vomiting, diarrhea, painful ulcers on legs & feet)
4. ***Arthropods:*** animal with no internal spine, a body made of joined segments, and a hard covering, like a shell. EX: mosquitoes, ticks, flies are typical vectors for viral diseases (either mechanical or biological)

Eukaryote: Kingdom Protista (protozoa & algae) examples

1. ***Amoeba proteus:*** protist, used to be called Kingdom Protista. Animal-like protists = protozoa. In watery environments, extrudes pseudopods from cell which engulfs its prey. Free living; doesn’t cause disease.
2. ***Spirogyra:*** vegetative at far left, rest are conjugating. Have spiral chloroplasts. Filamentous algae having thin unbranched chains of cylindrical cells.

Prokaryotes: Domain Bacteria examples

1. ***Bacillus*** (rod-shaped) ***anthracis*** (anthrax): gram-positive, endospore-former, endospores can be weaponized for bioterrorism (resistant to heat, radiation, chemicals); causes 90% fatal disease anthrax.
2. ***Neisseria gonorrhoeae*** in pus: gram-negative, cocci in neutrophiles (phagocytes). Disease: gonorrhea, sexually transmitted disease (STD)
3. ***Nostoc:*** cyanobacteria, fix nitrogen in heterocytes. Photosynthetic. Is green because it contains chlorophyl.

Viruses are?

acellular microorganisms (not composed of cells) consisting of proteins and genetic material (DNA or RNA, but never both) that are inert outside of a host organism

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EX: Influenza, HIV, ebola

Prions are?

acellular proteinaceous infectious particles. Alpha-helix (normal) or B-sheet (rogue)

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EX: TSE (Transmissible Spongiform Encephalopathy) Mad Cow Disease, Kuru, CJD, Scrapie

Major taxa

domain, kingdom, phylum, class, order, family, genus, species

Is an arthropod vector involved in the transmission of the pathogen?

Yes, injection into the bloodstream of the host by the bite of an infected arthropod species like mosquitoes or ticks.

Cell morphology (shape)

1. sphere = coccus/cocci
2. rod = bacillus/bacilli
3. spiral = spirillum/spirilla (rigid) or spirochaete/spirochaetes ( flexibile)
4. endospore-containing rods *(Bacillus, Clostridium)*

**COCCI:** Cell arrangement (groupings): arise from cells failing to separate after division

Diplococci (2), streptococci (6), tetrad, sarcinae (4, 8), staphylococci

**BACILLI:** Cell arrangement (groupings): arise from cells failing to separate after division

Limited arrangements; bacillus, coccobacillus, diplobacillus, streptobacillus

Three domains (based on rRNA sequence)

archaea, bacteria, eukarya

Whittaker 5 Kingdom System

Monera (prokaryotes), Protists (formerly K. Protista), Plantae, Fungi, Animalia

*Saccharomyces cerevisiae* is also known as ____ yeast.

Brewer’s

Asexual reproduction is when?

1 mycelium produces spores via mitosis OR yeasts produce daughter cells via mitosis or budding

Sexual reproduction is when?

Mycelial cells of **opposite mating types** fuse → nuclei fuse → spores are produced via meiosis

Ascomycetes: showing asexual reproduction of?

*Saccharomyces cerevisiae*

Ascomycetes: conidiophore with?

conidia

Which ascomycetes is this?

*Claviceps purpurea* perithecium

Ascomycetes: This is showing *Saccharomyces* asci with?

ascospores

What fungi is this showing?

Basidiomycetes

Characteristics: Division **Ascomycota** “sac fungi” septate

1. Sexual reproduction: Ascospores in asci
2. Sexual structure: Ascocarps: morels, truffles, perithecial head (*C. purpurea*)
3. Asexual structure: Conidia
4. Disease causing member: *Claviceps purpurea, Candida albicans*
5. Disease: ergotism, thrush, vaginitis

Characteristics: Division **Basidiomycota** “club fungi”, septate

1. Sexual reproduction: Basidiospores in basidia
2. Sexual structure: Basidiocarps = mushrooms; *Agaricus bisporus*
3. Asexual: Conidia
4. Disease-causing member: *Amanita phalloides, Filobasidiella* (formerly *Cryptococcus neoformans*)
5. Disease: Mycotoxicosis, meningoencephalitis in HIV patients

Characteristics: Division **Zygomycota** “bread molds” aseptate

1. Sexual reproduction: zygospores in zygosporangia
2. Sexual structure: Zygosporangium, *Rhizopus*
3. Asexual: Sporangia
4. Disease-causing member: *Rhizopus, Mucor*
5. Diseases: bread mold, zygomycosis, mycosis in immunocompromised patients

Division Deuteromycota “imperfect fungi” - septate

1. Sexual reproduction: None; Ascospores or Basidiospores when found
2. Sexual structure: None found; asci or basidia when found
3. Asexual: Conidia - *Penicillium* → Ascomycota
4. No disease-causing member or disease.