Lab Exam #1

Environmental Cultures

• Classifications: normal flora, pathogen, and subcategories

  • Normal flora(biota): normally live in or on the human body and don’t normally cause disease

    • Resident flora: always present, thrive, and reproduce successfully; can quickly reestablish themselves after hand washing or hand sanitizers

    • Transient flora: temporary inhabitants that arrive through the air or fomite(an inanimate object that can carry and transmit infection); does not quickly reestablish

  • Pathogens: microbes that cause disease

    • Opportunistic pathogens (opportunists) become pathogenic under right conditions

• Properties of agar: non-nutrient, hysteresis temperatures

  • 0%(liquid) ——— 1.5%(solid); in between is semi-solid

  • Agar is carbohydrate derived from algae but provides no nutrients for microbes

  • Temperature hysteresis: melts/liquid at 85°C but solidifies at 40°C

• Identify and describe media types: broth, plate, slant, deep

  Medium	Nutrient Suspension	Container
  Broth	Liquid	Test tube
  Plate	Solid	Dish
  Slant	Solid	Test tube
  Deep	Solid-semi-solid	Test tube

• Identify and describe macroscopic growth: colony, lawn, pellet, pellicle, turbidity

  Macroscopic Growth	Medium	Description
  Colony	Plate	Distinct, visibe mass of cells that arose from a single cell; all cells are of a single species
  Lawn	Plate	Continuous growth on agar’s surface; confluent lawn
  Pellet	Test tube	Mass of growth at bottom
  Pellicle	Test tube	Mass of growth floating at surface
  Turbidity	Test tube	Cloudiness growth (NG – +4)

• Define binary fission

  • binary fission: bacteria reproduction; one cell splits into two —> each of those cells splits into two (doubling population with each instance)

Nasal Culture

• Define “pathogenic” and “virulence”

  • pathogenic: disease causing

  • virulence: severity of disease

• Functions of BAP

  • Blood Agar Plate (BAP): agar plate added with 5% sheep blood —> added to provide extra nutrients to encourage growth of picky (fastidious) bacteria

  • can help detect hemolysins (enzymes) that damage red blood cells

• Analysis and definition of hemolysis types

  Hemolysis result	Effect on RBC	Appearance on BAP
  Alpha	Partial lysis	green/brown around growth
  Beta	Complete lysis	Transparent halo
  Gamma	No effect	Media unchanged

Hand Washing

• Principles/ingredients in soaps, antibacterial soaps, hand sanitizers

  • soaps/detergents: not designed to kill microbes, acts as surfactants —> reduce surface tension so its easier to lift dirt, oil, grime & microbes

  • antibacterial soaps: acts as surfactants but have chemicals that target microbial cell components to kill them

    • chlorine based substances that disrupt cell membranes

    • triclosan was a common ingredient

  • hand sanitizers: alcohol based; denature proteins and disrupt cell membranes

    • must be 70% isopropanol or 60% ethanol

Aseptic, Streak Plate Technique

• Terms: medium, sterile, mixed culture, pure culture, contamination, aseptic,
  nonchromogenic

  • medium: (p. media) nutrient suspension —liquid, solid, semi-solid— within a container

  • sterile: free of living things

  • mixed culture: a medium that contains two or species of microbes

  • pure culture: medium that contains only one species of microbes

  • contamination: accidental introduction of unwanted microbes

  • aseptic: free from contamination

  • nonchromogenic: white, gray, beige colonies; also called “uncolored” or “nonpigmented”

• How to label plates

  • student name, date, group number, & microbe of culture (if known) or source of sample

  • along the periphery of plate to allow observation of growth

• Function of streak plate technique

  • Used to isolate bacteria from each other allowing easy subculture and production of pure culture

  • Sample is spread/streaked across quadrants; cell density from one quadrant to the next is decreased

Microscopy

• Microscope parts and their functions

• Calculate total magnification

  • Total magnification = mag. of current objective X eyepiece mag.

• Terms/concepts: working distance, field of view, depth of field, resolution, brightness

• Purpose of immersion oil: refractive index

  • Oil immersion lens has the smallest opening causing much of the light to be lost and a blurry image→ must use immersion oil which has the same density/refractive index of glass slide that reduces the refraction and lost of light

Wet Mount, Hanging Drop Slides, and Semi-solid tubes

• Flagellar arrangements

  Atrichous	Having no flagella (but other structures)
  Monotrichous	Single flagellum at one end only
  Lophotrichous	2+ but only at one end
  Amphitrichous	At least one at both ends
  Peritrichous	Many flagella around

• Brownian motion: movement due to collisions with surrounding molecules; swaying motion like ocean

• wet mount: involves adding a drop of culture onto a slide and then placing a coverslip on top of the drop

  • function: creates a thin sheet of fluid where microbes can freely move side to side but not up and down

• hanging drop method (depression slide): creates a drop of culture that is suspended from a coverslip and housed within a dimple of a depression slide

  • function: allows for greater depth of movement and longer observation

• semi-solid tube: contains 0.4% of agar rather than 1.5% of solid agar

  • analyze semi-solid tube result: results in non-motile microbes being suspended at original inoculation site while allowing motile microbes to move about; based on turbidity

• Compare advantages and disadvantages between these three methods

  method	pros	cons
  wet mount	quick, easy, & inexpensive	difficult to observe bc they can blend into background
  hanging drop	allows for greater depth and longer observation time	more prep & expensive
  semi-solid tube	safety	cannot observe movement during but only after with turbidity

Smear Prep, Simple Stain, & Negative Stain

• Identify bacterial structures: arrangement + shape

• Define chromophore, auxochrome; principles of how stains work

  • chromophore: part of a dye’s chemical structure that determines color

  • auxochrome: part of a dye’s chemical structure that determines whether it carries a positive or negative charge

    • cationic/basic dyes —> colors cell

    • anionic/acidic dyes —> uncolored cell

  • Principles:

    • Cells have a negatively charged cytoplasm

    • Opposite charges are attracted to each other

• Steps of a smear preparation, function of heat-fixing

  • Transfer: cells transferred from culture to slide

  • Spread: thinly spread a monolayer of cells

  • Dry: dry completely

  • Heat-Fix: pass over flame to kill organisms and adhere them to glass slide without protein denaturation

• Identify pictures of positive stain versus negative stain

  • positive = colored cells/uncolored background

  • negative = uncolored cells/colored background

• Compare/contrast simple stain procedures

  • direct: smear prep —> color —> rinse+dry —> observe

  • indirect: aseptic transfer —> color + push slide —> dry —> observe

Gram Stain

• differential stain (involves multiple reagents in multiple steps) that differentiates on cell wall structure —> gram-positive or gram-negative

• cell wall made of 3 components(s—>d):

  • Glycocalyx – outermost coating of sugar and proteins or lipids providing cell with

    protection against dehydration, endocytosis, antibiotics, etc § Facilitates cell adhesion and formation of biofilms

    • Biofilms – community of microbes that come together for survival

      • Loose and unorganized glycocalyx – slime layers

      • Gelatinized and well-organized and firmly attached to cell – capsules

  • cell wall – made of carbs, proteins and lipids that play a role in cell shape and structure

    • deep to glycocalyx

    • important component – peptidoglycan

      • the thickness will determine whether bacteria are gram positive or gram negative

      • gram positive – 2-3 time thicker than gram negative

      • gram negative – includes secondary superficial membrane called

        the outer membrane

  • cell membrane – selectively permeable barrier that is between inside and outside

    • of cell that allows what substances can pass

      • deep to cell wall

        Primary dye	Crystal violet	1 min Gentle rinse F: stain all cells	Purple	Purple
        Mordant	Gram’s iodine CVI = crystal violet iodine	1 min Gentle rinse F: bind with CV = CVI complexàincreasing CV affinity to peptidoglycan	Purple	Purple
        Decolorizer	50% acetone/alcohol	3-8 secs ~ 5 sec avg Gentle rinse entire slide F: dissolves outer membrane and strip CVI complex from peptido	Purple	Colorless
        Secondary dye	Safranin	2 mins F: counter stain with red	Purple	Red

• Troubleshooting

  • Adjust decolorizer

    • Too short = false +

    • Too long = false – o Adjust smear thickness

  • Too thick = longer decolorizing time and viced versa if too thin o Culture age = 24 hrs to several days

• Analyze Gram stain result: identify reaction+shape

  • Gram positive – purple

  • Gram negative – red/pink

• In class experiment

  • S. aureus – gram positive cocci

  • E. coli – gram negative rods/bacilli

  • B. cereus – gram positive rods/bacilli

Acid Fast Stain

• Identify acid-fast stain

  • Differential stain used to identify species of mycobacterium aka acid fast bacilli

  • Used for acid fast bacteria that have unique cell wall structures that hinder uptake of water based dyes that are used in gram’s stain

• Major cell wall component of acid-fast organisms

  • Has peptidoglycan but mycolic acid – waxy fatty acid is predominate component

  • Mycolic acid

    • Increases bacteria resistance to drugs, antibiotics and phagocytosis and dehydration which is difficult for gram stain

• Genus/species and diseases diagnosed by acid-fast

  • Mycobacterium genus

    • mycobacterium tuberculosis - tuberculosis

    • mycobacterium leprae – leprosy

      Dye	Specific	Steps	Acid fast (+)	Non acid fast (–)
      Primary dye	Carbol fuchsin	5 min Phenol which can dye cell and mycolic acid	Purple/pink	Purple/pink
      Decolorizer	Acid alcohol	10-30 sec F: strops carbon fuchsin from non acid fast cells	Purple/pink	Colorless
      Secondary dye	Loeffler’s methylene blue	2 min F: colors cells Decolorized cell non acid fast cells – blue color	Purple/pink	Blue

• Analyze acid-fast stain results

  Acid fast positive	Bright pink/red	Bacilli or spindle shaped (tapered)
  Acid fast negative	Light blue	Clumped together

• in-lab experiment:

  • M. leuteus – blue cocci – tetradcocci

  • M. smegmatis – purple rods

Endspore Stain

• Identify an endospore stain

  • Differential stain that is used to identify species that produce spores

    • These bacteria can initiate sporogenesis aka sporulation and modify their cellular structure to produce highly resistant structures

• Define the spore coat and dipicolinic acid

  • Spore coat – tough outer protective coating that protects spores from chemicals,

    mechanical disruption, etc. and produces dipicolinic acid

  • Dipicolinic acid – binds to and increases bacteria, DNA’s resistance to heat

• Species that produce endospores

  • Clostridium

    • C. tetani – muscle tightening

    • C. botulinum – muscle paralysis

    • C. difficile – colon inflammation

  • Bacillus

    • B. anthracis – skin, lung, digestive function

    • B. cereus – food born infection

      Dye	Specific	Steps	Spores +	Vegetative cells (spore – )
      Primary dye	Malachite green	5 mins off and on Heated into smear to penetrate outer coating of spore	green	green
      Decolorizer	water	Sufficient amount of water to remove dye from vegetative cells	Green w/clear bubble	colorles
      Secondary dye	Safranin	2 min F: counterstain delcolorized cell dyed

• Analyze endospore stain result

  • Vegetative cells – red

  • Endospores – red bubble and green

  • Exospores – green

    • Exospores look like well-defined bacillus or sesame seed like shape

  • Green colored spores (endo or exo) = positive result

Capsule Stain

• Identify a capsule stain: vegetative cell vs capsule color

  • Different stain that allows to view bacterial capsule

    • But because non-ionic are poorly stained

    • Must follow stain by negative stain followed by modified positive stain that will stain everything but bacterial capsule

    • Capsule color will appear as halo around vegetative cell

  • Vegetative cell – bacteria that are metabolically active and reproduce normally 3 main steps

    • No heat fixing step of positive stain because capsule is delicate so chemical fix instead

  • Chemical will fix and kill and adhere organism to slide but wont dehydrate or deform capsule

• Steps

  • Indirect – negative stain – 1% Congo red

    • Aseptically transfer sample OFF CENTER § Add 1 small drop Congo red

    • Push smear

    • Dry completely

  • Chemical fix – 0.1 N HCl

    • Gently cover entire slide with HCl and sit for 30 sec

    • Pour off excess and dry completely NO RINSE

  • Positive stain – safranin

    • Safranin – 4 mins

    • Gentle rinse and dry

      Dye	Steps	Capsule +	Capsule -
      Congo red	Impart red color	Red background	Red background
      HCl	Chemically fix	Blue background	Blue background
      Safranin	4 min Apply to smear to impart stain to vegetative cell	Blue background red capsule w/ clear halo	Blue background Red capsule

Controlling Microbial Growth

• Growth curve - graphical representation of the change in a population over time and has four main phases

  • Lag phase:

    •  period of adjustment immediately after being introduced to a new

      environment

    • DNA replication and protein synthesis are taking place

    • rate of binary fission is low so no significant change to populationas

  • exponential growth (log phase):

    • cells undergo the highest rate of binary fission producing a rapid growth in the population

    • will continue as long as nutrients are plentiful and waste accumulation is low

    • birth > death

  • stationary phase:

    • growth slows due to decreased nutrients

    • increased population density—>waste accumulation causing population growth to plateau

    • birth = death

  • death phase (decline phase):

    • lack of nutrients and waste accumulation to toxic levels causing reduction in viable cell number

    • birth < death

• Define:

  • Static – substances or conditions that inhibit growth of cells without killing them

    • Termed based on organisms targets

      • Ex. bacteriostatic, virostatic, and fungistatic – bacteria, virus, fungi

  • Cidal – substance or conditions that kill cell

    • Termed

      • bactericide, virucide, and fungicide

  • Disinfect – substances or conditions that reduce number of microbes but often have poor effect on bacterial spores

  • Sterilize – sterilizing effect = all viable cells and spores are destroyed

Osmotic Pressure

• Define
  o Solute –substances that are dissolved
  o Solvent – water
  o Solution – homogenous mixture of one or more solutes that are dissolved in solvent
  o Plasmolysis - water drawn away from the bacterial cell—>causing cytoplasm to shrink away from cell wall

• Define:

  • Tonicity – how water moves bw two solutions separated by membrane

    • Isotonic – no net movement of water = equal

      • Same solute [ ] in and out

    • Environment/ solution

      • Hypotonic soln – low solute [ ] and low osmotic pressure—>excess water—> water will move away and inside of cell (hyper) – cell lyse

      • Hypertonic soln – high solute [ ] and high osmotic pressure—>

        lacks water—>water will move out of cell (cell = hypo) – cell crenate

• Analyze osmotic pressure result

  • Osmosis - movement of water across semipermeable membrane to achieve chemical balance, water moves from high water concentration towards low water concentration

  • osmotic pressure: the degree to which osmosis is occurring (the greater the concentration difference between two solutions, the greater the osmotic pressure

  • osmophiles – microbes that prefer high osmotic pressure

    • —>hypotonic environment limits there growth

    • Halophilic - “salt-loving” microbes—>require higher salt concentrations to survive

    • Saccharophilic - “sugar-loving” microbes—>require higher concentration of sugar for survival

Temperature, Moist Heat Methods

• Define:

  • psychrophile: 0 °C to 20 °C

  • mesophile: 20 °C to 45 °C

  • thermophile: 45 °C to 80 °C

  • subcategories

    • psychrotolerant organismsa—>prefer the mesophilic ranges but can grow slowly at refrigeration temperatures, approximately 4°C

    • hyperthermophiles aka extreme thermophiles—>prefer temperatures of above 80 °C up to as much as 113 °C

  • Analyze temperature results

    • temperature has a direct effect on the amount of energy a chemical reaction has and the rate of successful collisions

    • lowering temperature—>slows the activity of all molecules, decreasing reaction rate

    • raising temperature within a certain range optimum—>increases activity and increases the reaction rate

    • raising above an organism’s optimum—>denature (unfold) enzymes and inactivate them

    • exp results

      • staph aureus—>37 °C—>mesophile

      • e. coli—>37 °C—>mesophile

      • b. stearothermophilos—>42 °C—>mesophile

      • p. fluorscens—>25 °C—>mesophile

• Analyze moist heat method results (disinfect vs sterilizing)

  • o Moist heat vs dry heat

    • dry heat: flaming with a Bunsen burner or your gas or electric oven at home

    • moist heat: boiling or steam

      • results in more efficient transfer and penetration of heatàto

        achieve the same effect of microbial control as dry heat but at

        lower temperatures

    • disinfect – kill many microbes excluding spores

    • sterilize – KILL ALL microbes including endospores

• Know moist heat method conditions:

  • boiling water: 100°C, > 1 minute; disinfect

    • most simple and common method among campers and hikers to make water drinkable

      • recommends holding water @ rolling boil (100°C) for at least 1 minute (more for higher altitudes)

  • pasteurization: 63°C, 30 minutes; disinfect

    • aims to reduce the number of microbes in food and drink w/o

      compromising its taste and texture

    • many different methods and depends on the consumable product and the industry—>all aim to disinfect

    • lab implements the ‘low temperature long time’ or LTLT = 63°C for 30 minutes

      minute (more for higher altitudes)

  • autoclave: 121°C at 15 psi, 15 minutes; sterilize

    • steam heated to 121 °C is injected into a vacuumed chamber

    • pressurized to 15psi (normal is about 14.7 psi) and held at this condition for at least 15 minutes

  • tyndallization: 100°C, 15-30minutes, incubate and repeat > 3 times; sterilize

    • simpler but more time-consuming process

      • subjecting substance 100°C for at least 15 mins then allowing to rest or incubate x3 times

    • resting in-between period allows spores present in the substance to germinate à resulting vegetative cells are then neutralized in the following heating period

Oxygen Requirement

• Define classifications:

  • Aerobe – requires o2

  • Anaerobe – doesn’t require o2

  • Subclasses

    • Aerobes

      • obligate aerobes - must have O2

      • microaerophile - require low O2, high carbon dioxide (CO2)

    • Anaerobes

      • obligate anaerobes - harmed by O2

      • facultative anaerobes - prefer O2 but can live without

      • aerotolerant anaerobes - unaffected by O2

• Identify and understand

  • candle jar – c jar—>burning wax reduces O2 levels while producing CO2 and water vapor (H2O(g))

  • anaerobic jar – a jar—>AnaeroPak system consists of a container and a pouch that produces CO2 and hydrogen gas (H2) once exposes to the atmosphere.

    • H2 combines with O2 to produce H2O(g).

    • A jar is incubated with an indicator pill which should remain pink with a properly working system —> turns purples when exposed to O2

  • thioglycollate medium

    • sodium thioglycollate deep: sodium thyioglycollate reduces O2 to H2O

      • The medium is a semi-solid agar—>slows diffusion of O2 from the surface and allows sodium thioglycollate to completely reduce O2 deeper in the medium.

      • contains the O2 indicator resazurin, which turns pink where O2 is present but colorless where O2 is absent.

• Analyze oxygen requirement results

  • M. letues—>A jar 37 °C—>W—→gradient – more top less bottom—> facultative

  • E. coli —> A jar 37 °C —> W + —> gradient – more top less bottom —> facultative

  • S. aureus—>A jar 37 °C—>W+—>gradient – more top less bottom—> facultative

  • C. sporogenes—>A jar 37 °C—>W+—>gradient– growth below resazurin→ obligate anaerobe

pH

• Define terms and analyze pH results: acidophile vs neutrophile vs alkaliphile

  • acidophile: growth optimums at a pH of 2

  • neutrophile: growth optimums at a pH of 7

  • alkaliphile: growth optimums at a pH of 10

*use turbidity to determine growth (NG, +1,+2,+3)

Ultraviolet (UV) Light

• Identify UV experiment

• Define ionizing, non-ionizing

  • ionizing: ability to remove electrons from atoms thereby creating ions; shorter wavelengths that carry more energy

    • ex: X-rays and gamma rays

  • non-ionizing: unable to create ions, but still has enough energy to excite electrons and affect bonds between atoms; longer wavelengths that carry less energy

    • ex: microwaves, visible light, radio waves, UV light

• Define UVA, UVB, UVC, germicidal, thymine dimer

• germicidal: ability to destroy/kill microorganisms

  • UV-C is germicidal 

• thymine dimer: most common pyrimidine dimer, and accumulation disrupts DNA replication and transcription

Agar Diffusion, Kirby Bauer

• Identify Kirby Bauer method, medium used for Kirby Bauer

  • Kirby-Baurer Method: FDA-approved agar diffusion method that addresses variabilities in regular agar diffusion(excess nutrients in medium, larger/thicker plate, increased turbidity of microbial sample, and ext. incubation periods)

    • uses Mueller-Hinton agar plate 

• Subculturing to determine effect

  • subculture a sample from the ZOI→if no growth means antimicrobial agent has cidal effect; growth means agent has a static effect

• Analyze Kirby Bauer result: 

  • resistant: microbes that are not significantly affected by agent

  • intermediate: higher dose is required or may need to be used in combination with other drugs to achieve sufficient effect

  • susceptible:microbes that are negatively affected by/sensitive to an agent

  • cidal: substance/condition kills the cell

  • static: substance/condition that inhibit growth of cell without killing them 

• Define: 

  • antibiotic: substances naturally produced by an organism to inhibit other organisms

  • semisynthetic: antibodies that have been modified in the lab to increase effectiveness or to overcome microbial defenses

  • synthetic: chemicals (ex. dyes) developed in labs and found to have antimicrobial function

  • broad spectrum: effective against a wide range of microbial species; example can affect both gram +/-

  • narrow spectrum: effective against a very limited number of microbial species; example can affect only gram(+) or only gram(–)

Viruses: Bacteriophage

• Define: 

  • specificity: only able to infect a particular organism; determined by surface protein receptors on both virus and host

  • complementary: similar binding shape

  • virulent virus: replicate and release new virions through immediate host cell destruction

  • temperate virus: may cause immediate host cell destruction but have the additional option of integrating viral DNA into host DNA and lying dormant

  • lytic cycle: phage replicates and lyses host cell

  • lysogenic cycle: phage DNA incorporates into host DNA; replicates whenever host does 

  • plaques: clear zones resulting from an infected bacteria that releases new phages and continues cycle

• Identify T4 bacteriophage structures

  • protein capsid→houses viral DNA

  • contractile sheath

  • base plate→produces lysozyme and allows for the injection of viral DNA through E. coli cell wall

  • tail fibers→serves as receptors for specific E. coli binding 

• Understand cultivation of bacteriophage

  • cultivate (grow) to determine their titer (concentration, number of viruses) in a sample

  • undiluted sample will contain trillions of virions so we must do a serial dilution to simplify calculations

    • stepwise reduction of concentration with host E. coli

      • confluent lawn created→incubation→plaques formed by infected E. coli that release new phages and continue cycle 

      • only plates with 30-300 plaques are used for titer calculation 

      • each plaque=plaque forming unit(PFU)  

• You are not responsible for calculating titers

Kingdom Fungi

• Identify thallus and microscopic structures: Aspergillus, Penicillium, Trichothecium, Rhizopus, Rhizopus zygospores, Saccharomyces cerevisiae, conidia/conidiospores

• Identify microscopic structures: Schizosaccharomyces spp., ascus, ascospores

  • single mother cell is the ascus and the clusters inside are the ascopores

• Know the function of SAB

  • reduce contamination of fungal cultures by bacteria via SAB (sabouraud dextrose agar)

  • inhibits bacterial growth through its high dextrose concentration (high osmotic pressure) and low pH of 5.6

Kingdom Protista: Protozoan

• Identify pictures of protozoan species and their morphologies (trophozoite, cyst, schizont, ring form, where applicable): Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma gambiense, Balantidium coli, Toxoplasma gondii, Plasmodium falciparum

• Identify disease and transmission for each protozoan species