Microbiology: Metric System, Solutions, and Dilution

Metric System in Microbiology

• Scientists utilize grams for mass, meters for length, and liters for volume in measurements.
• Parts of cells measured via microscope are in micrometers (µm), where 1 mm equals 1000 µm.
  • Structures too small for micrometers are measured in angstroms (Å), 1 microliter is equivalent to 10,000 Å.

Solution Concentration and Dilution

• Solution concentration is the measure of solute particles in a solvent.
• A solution can be a mix of two or more substances where molecules are evenly distributed.
• Solution concentrations are defined by the molar concentration of chemicals.
• The 'X' system involves stock solutions (e.g., 10x or 100x) diluted before experiments.
• Percent system expresses concentration, for example, 10% of 100 ml solution involves using 10 ml diluted to 100 ml.
  • Using powdered reagent means solution is weight per volume, with 1 ml of water weighing 1 g.
• Microorganism populations may require dilution for enumeration of individual colonies.
  • Dilution involves pipetting a sample into a diluent like sterile medium organism was in or distilled isotopic saline (0.85% NaCl).
  • Dilution factor equals sample volume divided by total volume (sample + diluent volume).
• Serial dilution is useful by doing the same dilution repeatedly in a series of tubes, especially for small volumes of very dilute material.
  • Series of smaller dilutions yields greatest accuracy when very large dilutions are needed, as statistically determined.

Common Microbiology Tools

• Unique inoculating tools include serological pipettes (bulb needed), Pasteur pipettes (suction bulb), disposable transfer pipettes (non-graduated) and inoculating loops/needles.

Sterilization Techniques

• Sterilization eliminates all living microorganisms, including vegetative cells and spores.
• Autoclaves use moist-heat sterilization, producing high-pressure steam for high temperatures.
  • Temperatures upwards of 121°C for at least 15 minutes kill everything, including endospores.
• Membrane filters are used when tools cannot withstand extreme heat, physically removing all microorganisms.
  • Made commonly of cellulose acetate or polycarbonate in varying pore sizes, passing solution through 0.45 µm (removes most) or 0.22 µm (if for ultramicrobacteria or viruses) assures sterility.
• Bunsen burners employ incineration, the most common sterilization method.
  • The hottest part of the flame, the inner cone, sterilizes, while the outer cone heat-fixes smears and incinerates open glassware mouths.
    • Correct the flame by making it smaller with the knob if the inner cone isn't visible.
    • Depressing and rotating the touch pad until it locks down relights the flame, and a flint striker lights the burner.
  • Flames provide a radial sterile field by creating an updraft, pushing away bacteria and minimizing contamination using aseptic techniques.

Types of Laboratory Tubes

• Snap cap, Pyrex glass, screw cap conical, and microcentrifuge tubes are common laboratory items.
  • Most tubes are plastic, except Pyrex glass.

Measuring and Controlling Temperature

• Temperature reflects the average kinetic energy of a substance's molecules.
• Recommended overnight equilibration of apparatus in water baths or incubators for temperature control, with cultures ideally incubated at 37°C for optimal growth.

Spontaneous Generation of Microbes

• The idea of life arising from nonliving things existed into the 17th century.
• Louis Pasteur knew air contained fermentation-spoiling microbes, developing pasteurization to heat beer wort or wine juice and kill them.
  • Doubting spontaneous generation, he filtered air through guncotton and saw microbes under a microscope.
  • Swan-neck flasks were heated to kill microbes, the narrow bent neck allowed air entry but trapped microbes.
  • Microbes didn't grow until the flask neck was broken, showing microbes came from the air, not spontaneous generation.

Microorganisms & Their Environment: Culture Manipulation

• Ubiquitous microorganisms commonly pose no threat and can be beneficial to healthy humans.
  • Normal microbiota act as the body's initial defense, competing with invading pathogens, but opportunistic microorganisms can thrive if they are not present.
• Soil organisms play roles like processing phosphorus and nitrogen, aiding other organisms, but spores of tetanus and botulism bacteria can be harmful.
• "Pure cultures," containing one organism type, allow colonial and cellular morphology study to distinguish traits for identification.

Streak-for-Isolation Technique

• A technique using inoculating loops to dilute out organisms onto the surface of a nutrient agar plate.
  • Liquid, semi-solid, or solid medium is used.
  • Petri dishes have solid medium/agar, tubes have nutrient broth (NB).
  • This separates organisms in a sample by achieving the smallest possible population of organisms in the plate's center.

Isolated Colony Definition

• A single isolated colony is a population of bacteria or mold that comes from a single deposited cell.
  • Nutrients enable microbial cell division and growth into macroscopic colonies.

Liquid Broth Growth

• Growth in liquid broths can be seen through cloudiness or turbidity distributed throughout, or concentrated at the top or bottom.
  • Visible turbidity in a culture indicates around 10^6 microorganisms per milliliter.

Inoculating Devices

• Inoculating loops and needles are made of nichrome.
  • Because microorganisms are ubiquitous, the loop is passed through the top of inner cone, turning red, and the wire is sterilized.
    • Instruments are also able to be incinerated.
  • Wires must always be sterilized before and after cultures are transferred to prevent carryover, environmental contaminants, or surroundings contamination.

Subculturing Definition

• This is when microorganisms transfer from one medium to another.

Experiment Procedure: TSA Plate

• A TSA plate and nutrient broth are inoculated using two sampling sources, one from the body and another from the environment.
• The swab needs to be wetted first.
  • Never place the agar plate face-up on the bench to streak, to avoid air contamination.
  • Hold the plate and partially open the lid for streaking.
• Pass the tube lip through the flame when using broth.
• Plates should be incubated upside down, which stops water condensation from disrupting developing colonies dripping onto agar surface.

Analysis of Bacterial Colony Morphologies

• Configurations: round, complex, filamentous, wrinkled, concentric, rhizoid, lenticular (embedded in agar), irregular, and spreading.
• Elevations: flat, raised, convex, pulvinate, umbonate, hilly, ingrowing, crateriform.
• Margins: entire, wavy, lobate, irregular, filamentous.
• Isolated colony descriptions should include:
  • Color (yellow, white, colorless, opaque, etc.)
  • Size (diameter in mm)
  • Configuration, Texture (shiny, dull, mucoid, hairy)
  • Margin or edge, and Elevation
• Growth patterns in nutrient broth/liquid media can be diverse.
• Thin layer - pellicle, thick layer - membrane
• Large clusters of organisms show a flocculent growth pattern distributed throughout the medium.
• Uniformly turbid, flakey, or granular are possible below surface growth. Sediment can be gently agitated to determine sediment. Additionally
  • Turbid means growth.
  • A ring, pellicle, or membranous flocculent can grow on the surface.
  • Flakey, sediment, granular can grow below.

Light Microscopy

• Light microscopes are compound microscopes from their many lenses.
• Bright field microscopy uses light passing through specimen for illumination.
• Magnification: magnifying the image of an object on film (10X by the ocular/eye piece lens).
  • Magnifies further from 4X, 10X, 40X and 100X using objective lenses
• Total magnification multiplies ocular by objective lens magnification, with a max of 1000X.
• Condenser lenses, below the stage, focus and align light on the specimen being studied.

Controlling Light in Microscopy

• An aperture iris diaphragm regulates the amount of light passing through the condenser lens, which controls contrast.
• At the microscope's base, the field iris diaphragm controls the area of illumination, with knob controlling size.
• The depth of field is viewed from above, measured in µm, and decreases with magnification.
• Increased resolving power decreases the distance at which two image points can be distinguished.
• Microscope resolution is limited by the numerical aperture (NA) and the light's wavelength (λ) shown in the value 0.61λ / NA
  • Smaller wavelengths reduce resolution limit and increase resolving power.
• The refractive index is bending deviation as light rays pass from one medium to another, occurring where two media meet.
• Immersion oil prevents light ray bending or loss from specimen through glass to air, increasing the refractive index and resolving power.
  • Damaging the 100X lens by improperly removing the oil after use is a con.

Steps for Kohler lllumination

• With all elements in perfect alignment and the iris diaphragm adjusted to the best setting:
  • Begin with the 10x objective, and move the stage down away ampely from the objective lens with the coarse adjustment knob.
  • Ensure that bring stage and slide are close as possible to the10x objective and that the aperture iris diaphragm completely opened ally - Look through an ocular lens, and bring the stage down until in focus with the coarse adjustment knob. Fine tune with the fine focus.
  • reduce the size of the illuminated field by roatating the knurled ring the illuminating ring

Determination of Bacterial Motility

• Some bacteria are non-motile, displaying no directed movement
  • Brownian movement can be observed in an aqueous environment. This erratic movement isn't true motility, and is caused by randomly-interacting molecules.
• The function of flagella enables true bacterial motility shown best through flagellar staining, movement is also determined by stabbing a semi-solid nutrient agar medium in a straight line.
  • Turbidity spreading away from the stab line shows movement
  • Direct microscopic observation of broth cultures in wet mounts or hanging drops help see motility.

Hanging Drop Method

• Materials include depression slide, NB with culture, coverslips, vaseline, applicator sticks, and a revolving 10x or 40x microscope
• Vaseline lines the coverslip edges, culture in the center, depression slide on top, then inverted
• Temperature increases Brownian movement via thermal energy and viscous solutions slow motility by adding resistance. Adjusting controls the amount of light entering the microscope, enabling better visibility of microbes in the droplet
  • Increases droplet contrast by narrowing field, increasing movement observation.

Winogradsky Column

• The experiment exemplifies how microbes occupy precise microsites based on environmental and carbon/energy needs.
• Some microbes make metabolic byproducts that are consumed by others, and more food sources are added the microbial community in the pond mud
  • Shredded paper is carbon source/cellulose (electron donor); calcium carbonate yields carbon dioxide, and calcium sulfate gives sulfur (electron acceptor).
  • Indirect sunlight the column's sole energy source.
• Organic material promotes microbial growth, which then depletes O2 and creates a high-to-low oxygen gradient (top to bottom)
• At the very top, aeration still occurs due to the water preventing enough 02 diffusion through to replenish it

Bacterial Composition

• Near the top, aerobes and microaerophiles will grow, and strict anaerobes grow at the very bottom.
• H2S gradients (low at the top, high at the bottom) occur because sulfate-reducing bacteria use fermentation byproducts (organic acids and alcohols are a carbon source)
  • H2S forms (from that process where sulfate is instead of oxygen is the final electron acceptor)
  • Black areas indicates H2S reacted with iron in the mud, forming ferrous sulfide.

Microbial Behavior in Column

• After several weeks, the column displays a zonal look, reflecting photosynthetic bacteria's adaptation to a specific oxygen and H2S concentration.
  • Green sulfur bacteria grows just above the dark layer, and need photosynthesis
  • Next layer is purple nonsulfur bacteria that thrive in the rust colored layer.
  • Any H2S is converted to sulfate via sulfur oxidizing bacteria in the aerobic where they uses CO2 and are chemoautotrophs with material derived at mud/water point.
  • A water layer exists, where aquatic organisms use light for energy and release of (O2), (CO2) is removed resulting to the columns oxygen gradient.
  • It functions self controlled recycling and this ecosystem allows different microbes to perform roles and to exist.

Smears & Gram Stains

• Specimens need contrasting stains but cytoplasm is transparent, so stains must be simple.
• Stains, crystal violet or methylene blue, contain simple dye in a single process to spot microbes via microscope, because bacterial ( – ) and chromophores (+).
• Bacteria can be in various shapes: spherical (cocci, sing. coccus), rods (bacilli, sing. bacillus), and spirals (spirilla, sing. spirillum), and variations.

Bacterial Division and Stain Reaction

• How number of planes in which division occurs determines cell arrangement and determines identifiability.
• Spirillas are usually single cells, cocci or bacilli can be multicellular. Pairs of daughter cells after division are diplococcus and this also happens in bacilli producing diplobacilli
• Cells dividing in long chain formation means it exhibits streptococcus or streptobacillus for cell structure
• Division in 2D forms a tetrad and a cube shows 3D called sarcina. Grapelike clusters are staphylococcus.
• Differential staining can differentiate bacteria based on their ability to retain a particular stain, which needs two or three chemical reagents.
• The gram stain helps separate bacterias due to cell walls.
• Heat fix a smear of organism by primary stain, crystal violet, then, Gram's iodine stain

Gram Staining

• The most significant step is washing the stained smears with a decolorizing agent mostly 95% Isopropyl or ethyl alcohol.
  • After this a counter stain is added, safranin. Gram (+), minimal lipid content, and THICK PURPLE.
• Gram negative bacteria has thin Peptidoglycan w/ high abundance of Lipid content that allows counterstain to appear red or pink.
• Excessive alcohol, can allow to decolorize cells.
• Because of cell wall degradation older cultures may not stain very properly. Which results in gram variation do incorrect staining.
• A small drop should be spread on the surface of the slide.
• Heat Fix the smear with flame/Multiple quick passes. Flood with Crystal violet. Water rinse. Gram's Iodine; water rinse.
• Apply ethanol until almost clear then water rinse and Flood smear with Safranin, then, rinse.

Stains

• If there are too few bacteria, the smear needs to be prepared over.
• Heat Fix the smear w Multiple quick passes
• This is what heat fixing does. It Prevents sample being. Washing of slide and maintaining it's morphology
• This is what Overheating Disrupt the Morphology Can cause shape change or lys cell/ staining

Gram + vs Gram -

• (Gram +) Think peptidoglycan layer W/teichoic acids, less Lipid W no other membrane, more susceptible to antibiotics (Gram -)

Endospore

• Forming spore creates deadly infections. Such an Anoreobic are are (Clostridium & Bacillus. Which contains Anthracis that Causes that anthrax
• Metabolic Vegetative Inactive, resistant metabolically inactive spores
• When Conditions Become Unfavorable for vegetative cells and, usually due the source of lack a carbon then Sporogensis and they rise to grow, intracellular, endospore . It's Worsen then Impervious a released sport
• Has a spore made out of genetic material. Hot in the environment and what happens to the protein which literally makes it impossible to get through the cell.
• the Malachite staining forces the sport of green into it.
• Spores can't be decolorizing because their is a a low amount or the endoscopes is central and its shape. Is spherical but some can become swollen..
• If in a more for comfortable environment than active but less of the germinating cells can be lower than the endospores .

Prepare sample by Malachite for minutes using the sides of beaker

1. use Bacillus samples that very with their days and then boil water over the beaker to make the stain
2. remove and distilled water with safranin Distilled
3. Five day had a most sporing .

Acid-Fast Staining

• Acid Fast Staining is as Similar to Gram. High content. The cells is for Diagnostic
• In diagnostic the tuberculosis bacteria can also be what they are also not that can't be as well.
• There can also be cold stains. that what we have and they can penetrate lipid the wall.
• They can also not be D colorize. Non acid can DeColoriZed to stains like light blue
• E colis is also in the stains

Aerobes and Microbes

• If It tests the carbon's energy for it, it will depend on what the carbon source of nutrition you are getting at and so If you are phototroph then you will do what you use the light as your source .
• Then the Bacteria needs its Nitrogen's as it's trace. elements the of chemoautotrophs use or both
• Because they will simultanously get energy at the the same time. To measure bacteria, need to count the total numbers, indirect or spectrophotometer for the mass .

Spectrophotometer. Needs all the media selective

• selective test you should perform for both medias. Indirect also shows better that way. Two mediums test.
• 30 mins and every time you get sample it is for the cuvette medium and the to measure the number you're trying Zero for the spectrophotometer
• Multiply the numbers together for for the ml is equal to the colonies that 20 the dilution and its platting factor.

In plating factor

• The amount that accounts the measure of the colony to represent the colony's amount. So it makes the value on the whole .

Dilution the medium

• It contains the isotonic and then there for the time is is for the original has . When you use a little a 0. To create for The duplicate of the agar will is you can use sterile to also. As well

Selective Mediums

• differential used for Mixed.
• The media allows them to be distinguished
• Following a characteristic
• Can also inhibit the media or microbes.
• Allows others to grow at a selective time. What allows the appear to be different each other

In the test E coli agar: 1- E coli has a green lactose when it produces an acid 2- Kleb has slow pink when it produces an lactose. Salmonella's also can not survive as well

Mannitol and what turns a what happens to the different what's it made out of Anaerobic a and that the tube is used. Are Brewer is

Bacterioidis

• what happens to all of a them when? You divide streak which goes with the tubes what tubes a that they will be doing .
• Which also goes with the top .

Metabolic Not sufficient. To differentiate the bacteria, what needs to be done. How they test the mediums that is for enzymes 1- Then they may also only to just the macro molecules or what's it made of. The reactions do always take where for Hydrolyzed . Indicates they have done anything to the medium a is to tell it the products

Heavy Poly:

• Then its mixes it with linear , amylose what causes it. What happens to heavy ,must be by: Bacteria is the break for what does what ? Glucose and test.

Reagent iodine is used for the starches and e and b. Reactions How would is that a e and what happened to B on the other hand. And also is has to get. Lipid and ex and this is what they degraded for. Alcohol Lactose. Then into monosaccharides. The that what they would ferment

• Glucose carbon enzymes a and e Can not determine if they are what or what The Durham a a b and what ends b producing. As for the proteus and .ECOLI Alkaline for is the what produces an alkaline for to begin 1- That does a thing 2- A little bit a thing

The wine

• is where .

• When natural enzymes . . to all other with the acid what the the of it. It also make to test it you might mix sugar and which .

The testing bacteria the Enterobacteriaceae. Large means 4 pathogens You can test it and what causes it for , if you're a can't. Which water. It may also show Aeromonas. Make an liquid incubation. Let the to grow . If you will can test the different.

Explore the metric system in microbiology, focusing on units like grams, meters, and liters. Understand solution concentrations, molarity, and dilution techniques. Learn to calculate dilutions and work with percentage solutions for accurate microorganism enumeration.