Microbiology: Measurements, Concentration, and Dilution

Fundamentals in Microbiology & Metric System

• Measurements performed by scientists use grams (mass), meters (length), and liters (volume).
• Cellular parts seen via microscope are measured in micrometers (µm); 1000 µm equals 1 mm.
  • Structures too small for micrometers are measured in angstroms (Å); 10,000 Å equals 1 µm.

Concentration and Dilution

• Solution concentration measures the number of solute particles in the solvent.
• A solution is a mix of two or more substances with evenly distributed molecules.
• Solution concentrations equal the molar concentration of chemicals.
• 'X' system solutions, like buffers, are stock solutions (e.g., 10x or 100x greater) diluted before experiments.
• The percent system expresses concentration; 10% of 100 ml solution equals 10 ml diluted to 100 ml. If using a powdered reagent, the solution is measured by weight per volume, with 1 ml of water weighing 1 g.
• In microbiology, microorganism populations may need dilution to make enumeration possible
  • In a dilution, a sample is pipetted into a diluent (sterile medium where organism was grown or distilled isotopic saline 0.85% NaCl).
  • The diluting factor equals volume of sample/total volume (sample + diluent volume).
  • Serial dilution involves repeating the same dilution in a series of tubes, useful for small, very dilute material volumes. The process provides greatest accuracy.

Common Microbiology Tools

• Serological pipettes (require bulb), Pasteur pipettes (require suction bulb), non-graduated disposable transfer pipettes, and inoculating loops/needles.

Sterilization

• Completely eliminates living microorganisms (vegetative cells and spores).
• Autoclaves sterilize liquid media using steam and pressure.
  • At temperatures upwards of 121°C, pressure kills endospores.
• Membrane filters are used for tools unable to withstand extreme heat
  • Bacteriological filters physically remove all microorganisms. Filters are commonly made of cellulose acetate or polycarbonate and come in different pore sizes.
  • Sterility assurance requires solutions to pass through pores of 0.45 µm (removing most organisms) or 0.22 µm (removing ultramicrobacteria or viruses).
• Bunsen burners are a common sterilization method (incineration)
  • Sterilization occurs in the flame's hottest part (inner cone).
  • Heat fixing smears and incinerating mouths of open glassware occurs in the outer cone.
  • The flame is adjusted to be smaller with the knob if the inner cone isn't visible
  • Relighting requires depressing and rotating touchpad until it locks down.
  • The flame provides a radial sterile field via an updraft, minimizing contamination, which serves as the main concept of aseptic techniques.
  • A flint striker is ideal for lighting a Bunsen burner

Types of Tubes

• Snap cap, pyrex glass, screw cap conical, and microcentrifuge tubes.
  • All are made of plastic except pyrex glass.

Measuring/Controlling Temperature

• Temperature measures average kinetic energy of molecules making up a substance.
• Apparatus requires overnight equilibration to the correct temperature (water baths, incubators).
• Cultures are usually incubated at 37°C for ideal growth.

Spontaneous Generation of Microbes

• Belief in nonliving matter creating life lasted until the 17th century.
• Louis Pasteur knew air had microbes that could spoil fermentation.
  • He developed pasteurization by heating beer wort or wine juice to kill spoilage organisms.
  • In experiments doubting spontaneous generation, he filtered air quantities through guncotton to capture airborne microbes that were then observed aplenty under a microscope.
  • He heated swan neck flasks to kill present microbes without sealing them letting air in.
  • The flasks' narrow bent neck allowed air in and trapped microbes.
  • Microbes did not develop in these flasks, even after months or years until the neck was accidentally broken and in a few days microbes were abundant.

Microorganisms/Environment: Manipulation of Cultures

• Ubiquitous microorganisms are typically not harmful and can be beneficial.
  • Normal body microbiota defend against invading pathogens by competing for resources.
  • Opportunistic microorganisms can become established in their absence
• Soil organisms process phosphorus and nitrogen.
  • Some may harbor harmful spores of the tetanus and botulism bacteria.
• Identification involves “pure cultures (one organism type), colony and cell observation distinguishing biochemical characteristics.

Streak-for-Isolation Techniques

• Loops dilute out organisms onto nutrient agar surfaces that can be liquid, semi-solid, or solid.
  • Solid medium/agar is contained in a Petri dish.
  • Liquid medium is contained in a nutrient broth (NB) tube
• Isolation separates organisms in any sample by minimizing the organism population in the plate center.
• In liquid broths, growth can be cloudiness or turbidity throughout the tube or focused at the top or bottom.
  • Visible turbidity on a culture has approximately 10^6 microorganisms per ml.
• Inoculating loops or needles are made of nichrome.
  • As microorganisms are ubiquitous, electricity is used to sterilizes the inner cone, which turns red, and all the wire part should be sterilized.
• Electric incineration is used to sterilizes wires before and after transferring cultures to eliminate any environmental contaminants.

Experiment: Sample Source Inoculation

• Subculturing: transfer microorganisms from one medium to another
• Two samples (one from the body, one from the environment) are inoculated into a TSA plate and nutrient broth.
• The swab must initially be wetted first to prevent air contamination.
• The agar plate should not be placed face-up when streaking.
• With broth, run the tube lip through the flame.
  • Plates are later upside down to stop condensation from dripping down and disrupting developing colonies

Bacterial Colony Morphologies Analysis

• 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.
• Well isolated colonies
  • Color: yellow, white, colorless, opaque, etc.
  • Size: diameter in mm.
  • Configuration.
  • Texture: shiny, dull, mucoid, hairy.
  • Margin or edge.
  • Elevation.
• Growth in nutrient broth/liquid media is diverse.
• Thickness includes thin layer (pellicle) and thick layer (membrane)
• Flocculent growth distributes throughout medium representing large organism clusters.

Turbidity vs Surface Growth

• Below the surface growth may consistently have uniform turbidity, flakey, or granular forms.
  • If sediment is present, it can be gently agitated to determine sediment
• Turbid = growth.
  • Surface growths may be ring, membrane, or flocculent forms
• Below growths are flakey, sediment, granular

Light Microscopy

• AKA: "the compound microscope" due to the many lenses.
• Illumination of specimens occurs where light passes through it for bright field microscopy.
• Magnification is the measure of relative increase in image size and involves both ocular and objective lenses.
  • The ocular (eye piece) magnification is 10X.
  • The objective lenses further magnify from 4X, 10X, 40X, and 100X.
• Total magnification multiplies ocular and objective lens magnification.
  • Maximum magnification is 1000X when using
• The condenser lens, found below the stage, aligns and focuses light.

Parts of The Microscope

• The aperture iris diaphragm regulates light passing through the condenser lens to control contrast
• The field iris diaphragm at the base of the microscope manages illumination area using a knurled ring rotation around the iris diaphragm
• Depth of field is the consideration of objects as viewed from above, and is very short.
• Depth of field decreases as magnification increases.
• As resolving power increases, the distance between two image points decreases.
• A microscope's resolution limit depends on transmitted light wavelength, and the aperture of the system at 0.61λ / NA
  • Shorter wavelengths decrease the distance and resolution limit, thus increasing resolving power
• Refractive index is the measure of light ray bending deviation transmitted between mediums.
  • Deviation happens at the junction between media
• Immersion oil prevents light ray bending or loss as they transmit glass through the air. This increases the refractive index and increases resolving power.
  • There is risk damages lens if oil is not properly removed on the 100x lens after lenses after use

Kohler Illumination Adjustment (Steps)

• Ensures perfect alignment of all elements, and adjust the iris diaphragm to produce a correct aperture.
  • Start with 10x, turn the coarse knob to lower the stage until lens and stage have ample distance
  • Raise the stage and slide as high as possible to 10x; ensure the iris diaphragm is completely open
  • Looking through the ocular lens, using the course focus knob, slowly bring the stage down. Then use fine focus for adjustments
  • Reduce illumination field using the knurled ring
  • Focus the field diaphragm by gradually adjusting height control using the condenser. The focus should form into an octagon shape (halo between red and blue)
  • Open it with the knurled ring until its edge is just outside the field of view, fully open
• Only adjust fine focus between lens uses

Determination of Bacterial Motility

• Some bacteria are non-motile (no directed oriented movement)
• The erratic movement of organisms in an aqueous environment is brownian movement
  • Caused by cells randomly interacting with molecule

Bacterial Motility

• True bacterial motility occurs with flagella.
  • Staining can determine motility; though tedious and not all flagella-possessing bacteria are motile
  • Movement of bacteria is also determined by “stabbing” a semi-solid nutrient agar medium in a straight line inoculation, then detecting turbidity from organisms that travel away from the line. Can also be determined through direct microscopic observation, either wet mounted or hung dry.
  • The hanging drop method uses depression slides with culture in NB, coverslips, vaseline, applicator sticks, and a revolving ECHO microscope.
  • Vaseline lines edges of glass coverslip and brother culture which is added to the center and the depression slide is place on top and observed. Increased temperature can cause an rise in browian movement
• Increased temperature can cause an rise in browian movement that lowers resistance.
• Adjusting an aperture iris diaphragm assists control light volume in microscopes for superior microbial visibility.

Winogradsky Column

• Exemplifies how environmental tolerances drive microorganisms to occupy specific microsites.
  • Some microbes also depend on the metabolic byproducts to survive
  • Adding shredded source cellulose (e.g., shredded paper), calcium carbonate yields carbon dioxide, and calcium sulfate releases source sulfur (electron acceptor).
  • Indirect sunlight delivers organic molecules from the sole energy source.
• The addition of organic material accelerates rates of microbial growth, depleting oxygen levels in the process and resulting in gradient of high to low oxygen gradient from top to bottom of a mud sample.
• The very top remains aerated because of the slow diffusion rate of oxygen through the water.
• Aerobes and microaerophiles occupy near top levels, whereas anaerobes are strictly at the bottom.
• Sulfate reducing bacteria produce gradients of H2S at high levels at the top and low ones, and the bottom has byproducts like carbon which leads to them using sulfate rather then terminal oxygen acceptor that lead to a generation of high levels of Hs2

Self Controlled Recycling System

• Self-Controlled recycling system requires zonal columns that can be black since HS2 in there reacted with iron from ferrous sulfide
• The appearance after weeks on is due in to various concentration of HS2 and 02 through the Thriving pigment bacteria
• Anaerobic photosynthetic species such as the green bacteria will be on top because bacteria are well above the dark layers where as purple grows that has low tolerance for high temperature They reduce for use of co2 light energy and calcium and have Hs2
• Top on that one layer is where purple doesn't sulfur since it has ananaerboic conditions and they are photohetertrophs of fermentation acids from there car con source
• There any hydrogen that dissolves their there oxidation to sulfur by the bacterias these materials is there from co2 and the water has cyanobactieria and algea to realeze these molecules like oxygen

Smears and Preparing for The Gram Stain'

• specimens must contrast with the environment
• to visulize the transparent Cytoplasm a simple staining is enough.
• a Cell has a simple dye since Chromphores is positive and and a Bacteria is negative
• They are called morphology since they have spherial
• The arangment can vary the cell as well if they devide after which can identify if the cell is divided

Steps of Process

• At times the cell division and staining can alter
• This differs simple since it can differentiate based on the strain
• The grand stand devoles around shape isolation and shape
• can be postive or negative
• Apply crystal violent stain and deep the stain as much as possible then iodine stain or an mordant
• wash till can see smear
• If a lot of acholol is appplied it can ruin a cell so a counter stain is needed
• Since a gram is too old if it’s stored to long it can lose a lot
• There are variable but it is not from staining itself.
• Do two passes for the cells,
• Apply a rinse with a mix
• Flood it wate and then ioxide
• Apply ethanol as possible Then stain it safranin
• Since the sample can have different stains the organism can also be tough to distinguish
• So heat is applied to prevent it but if too high then it desorts
• The water has to be pure.

Chapter 8: Endospore Stain

• Spore forming bacteria can serious affect and can also cause
• In anaerobic geneus
• There in metabolically active states
• It can come from a bad sorcerers and can cause sporganis and new structures
• A round and with inpprivous layers of conditions which allow
• Compose of enimes heat
• The dormancy allows everhting for damage to the material can revive
• It is easy to spot the spores if there is a primary color and the cells are stain green which can cause spores
• After decolorzied thye need to be stain
• This method is from sheffrer fulton and may depend on the shape and sizes
• They may resist through germination and the mn can cause formation
• There three type And use malachite green for 3-5 min And flood for around five days and the cell can indentift

Lipid-Fast Stain

• Differs from grand strains.
• High lipid content resists that are simple since they are made aqueous
• Stain is from diagnostic process that is pathogenitc
• Ziehll -nestlen and kinyo can be cold stans
• Lipid surrable is used that is why there red

Bacteria and Nutrition and Growth

• Types and the need to amount for optimal growth can come from different source source of what the carbon of. If it is organic It is heterozygous if it is carbon they can be self but mostly.
• Bach have different traces and sources nitrogen of what they are
• If simultanelsy then the are
• This increase pop growth
• Measure the a viable microscope cells on some area.
• Ecoli and others may not grow well
• Slectivw media

Indirect

• There broth made from davisi
• One ml in transfer
• Control each time as needed on each of all samples
• Cfu is calculated
• Sample divied by divident which us how many ml what the percentage

Dilution

• Is usually sterile saline Injected at 10 fold Direct is not an option Add glass beads and make it not hard Different organism are used for diff. Different is just something has been aded for some functions Distingushed from categories Chemicals from a medium Allows different bacteria to

An overview of lab measurements, concentration explained in microbiology. This includes grams, meters, and liters, micrometers, and angstroms. Also covers solution concentration, molar concentration, and dilution processes.