Bacterial Staining and pH Concepts

• An experiment includes asking a question, developing a hypothesis based on knowledge and observations and designing an experiment to test the hypothesis.
• Variables must be considered when designing the experiment.

Variables

• Dependent variable is what is being measured or observed in response to the independent variable and can have more than one.
• Independent variable is limited to one experimental condition, which is manipulated in the experiment.

Control Experiment

• Control experiments allow you to see the difference in results where the independent variable is either omitted/held constant.
• Control experiments give a baseline to evaluate results and experimental process.
• If the control does not work, one cannot make any conclusions about the experiment.
• It is best to have a positive and a negative control in every experiment, if possible.
• Positive controls are similar to the experimental test but give a positive result based on previous experience and confirm the basic experimental conditions could produce a positive result.
• Negative controls give a negative result and demonstrate the base-line result when the test does not produce a measurable positive result.
• Negative control values are treated as a "background" value to subtract from test sample results.
• Example: to test effects of doses of a drug (independent variable) on symptoms/signs (dependent variable) of illness, one can set up controls.
• Negative control omits the drug, so the effects of the illness without the drug are visible.
• Positive control gives the drug in a concentration previously shown to limit or modify illness symptoms/signs, giving the expected effect on the illness from the drug.
• Independent variable administers different doses of the drug than the positive control to see how the drug on the illness is dependent on dosage.

Use and Care of the Microscope

• A compound light microscope- brightfield microscope is used
• Objects are dark with a light field (background), has little contrast, two lenses (eye/object), and requires a light source.
• Always carry with one hand on the arm and one hand under the base.
• The base is the bottom/to carry
• The stage holds the slide
• The arm is to carry
• The body/observation tube transmits the magnified image
• The light source is in the base
• The image is inverted
• The condenser lens focuses light into a cone to concentrate light on the slide
• Iris diaphragm controls the angle and size of the cone of light
• Objective lenses rotate and vary in magnification.
• Primary lenses magnify 4x, 10x, 40x, 100x, and 1000x
• The eyepiece/ocular lens magnifies the image 10x
• The highest power objective is used for bacteria.
• Coarse adjustment focuses low power objectives (4x & 10x)
• Fine adjustment focuses high power and oil immersion lenses
• The field of view is the area seen through the microscope
• Scanning is 4x
• Low power is 10x
• High-dry is 40x
• Oil immersion 100x must be used with immersion oil, or the resolution will be low
• Total magnification = ocular (10x) x objective lens
• Always start low
• Lenses are parfocal
• Numerical aperture measures the lens’s ability to gather light and resolve fine detail
• Resolving power is when longer wavelength and white light give lower resolution, while shorter wavelength and electrons give greater resolution
• Refractive index is the amount of light that bends between the objective lens and the slide and changes when using immersion oil.
• Light is refracted when it passes from glass to air; with immersion oil, light loss is minimized, and resolution is enhanced
• Immersion oil reduces refraction without it, light bends outward between the slide and objective lens
• With immersion oil, light is not lost because it is not bent (refracted) from hitting air between slide and objective lens
• A clear image forms at the focal point
• Curved lenses give multiple focal points (spherical aberration)
• Adjusting the iris diaphragm reduces spherical aberration (light rays around the edge are eliminated)
• Phase contrast microscopes have two sets of light rays

Specimen Observation

• Phase contrast microscopes allow contrast between the specimen and dark field
• Phase contrast microscopes are used for living, unstained microorganisms and allows one to see internal structures
• Normal flora is bacteria naturally found on bodies
• Transient bacteria are bacteria picked up by hands throughout the day
• Brownian movement is caused by molecules in an aqueous environment
• True motility is when microbes move from one position to another, changing direction, spinning, or rolling
• Cell Division
  • Eukaryotes are bigger, have a membrane-bound nucleus and nucleolus, have membrane-bound organelles, may have complex appendages (arms, legs, tails, etc), and have uneven cell division
  • Prokaryotes are smaller, and can be seen under lower power
  • Both eukaryotes and prokaryotes have cell division
  • Bacteria usually undergo even cell division (binary fission)
  • Wet mounts are fast, don't require much equipment, but observing true motility is difficult because the cells are smashed between the slide and the cover (2 pieces of glass)
  • Hanging drops allow one to see microbes in their natural 3D environment
  • Hanging drops allow motile organisms to move and the petroleum jelly seal reduces evaporation of the liquid
• Aseptic technique includes practices that keep from contaminating media.
  • For example, test tubes and culture media are only open to introduce/remove something
• Sterilized refers to being free of all life
  • Sterilization usually uses an autoclave that doesn't physically remove bacteria but kills it

Inoculation and Cultures

• Inoculate means to introduce bacteria into growth media
• Broth cultures allow large numbers of bacteria to grow in a small place
  • Broth is a liquid with nutrients that allows bacteria to grow
  • Broth is used to quickly grow bacteria in a small space with no competition
• Turbidity is cloudiness of the liquid
• Flocculent is floating chunks
• Sediment is cells that float to the bottom, seen when bacteria require low O2 (oxygen)
• Pellicle is a floating mat of bacteria/scum layer, seen when bacteria requires high O2 (oxygen)
• If bacteria needs lots of oxygen, it may form a pellicle and they can protect the bacteria
• Agar slants are test tubes with solid agar at an angle to allow solid growth of bacteria on a surface but they are easy to store and transport and they are used for pure cultures
• Agar deeps are test tubes with solid agar at the bottom
• Used with inoculating needle to test bacterial oxygen requirements and grow anaerobic bacteria (requiring less oxygen)
• Semisolid agar deeps are more jello-like/watery than agar deeps
• Used to determine bacteria motility with an inoculating needle
• Motile bacteria look like upside-down christmas trees and are able to swim away from the inoculation site, and may be used to test oxygen requirements.
• Inoculating loops are used to transfer bacteria to agar plates, broths, and slants
• Always flame the needle using a bunsen burner flame until it turns red
• Inoculating needles are used to transfer bacteria to agar deep
• Binary fission is even cell division
• Streak plate technique grows single colonies
• Bacterium is single (one bacterial cell)
• Bacteria is plural

Staining

• Lactococcus lactis is gram positive
• Bacillus bubtilis is gram positive
• Escherichia coli- gram negative
• Staining bacteria enhances contrast so the bacteria can be seen with better detail and resolution
• Simple stain stains all bacteria with one reagent
• Chromophore is a colored ion that can be positive, negative, or neutral, depending on the dye
  • Basic dye has chromophore that is a positive ion (cation)
  • Acidic dye has chromophore that is a negative ion (anion)
• Direct stain stains the bacteria
• Negative stain stains the background of the slide, leaving the bacteria in its original form
  • Colloidal (nigrosin) particles are too large to enter the negatively charged chromophore of cell
• Differential stains are where bacteria react differently to multiple reagents
• Structural stains are used to identify specific parts
• Smears are a thin film of bacterial cells on a slide
• Fix attaches bacteria to the slide
• Heat fix is when bacteria are fixed to the slide by passing it through a flame

Procedure for heat fix & direct staining (simple):

• Spread a thin bacterial culture over
• Dry in air
• Pass slide through flame to fix
• Flood slide with stain and rinse
• Blot dry
• Place a drop of oil on slide and view with oil immersion lens
• Chemical fix is when bacteria are fixed to the slide with 95% methanol for 1 min
• Autolysis is the rupture of the cell wall due to cellular enzymatic digestion
• Denature unfolds or changes the conformation of proteins, rendering them inactive
• Bacteria have cell walls with slightly negative charge, and would be attracted to dyes that have chromophore with positive ion (basic dye), creating a direct stain
• Methylene blue has a positive charge while the slight negative charge of bacteria allows methylene blue to stain the cell
• Gram stain is used to classify and identify bacteria as gram-negative or gram-positive by the color it appears
  • Pink/red = gram-negative
  • Blue/purple = gram-positive
• Process & reason for gram staining procedure:
  1. Apply primary stain (crystal violet) to stain all bacteria purple by entering the cell; this pigment is only retained by gram-positive bacteria by the END of procedure
  2. Apply mordant (grams iodine) to form a crystal-violet-iodine complex; the iodine binds to the crystal violet chromophores, making a larger molecule that gets trapped inside thick cell walls of gram positive bacteria, but can be washed out of gram-negative bacteria
  3. Apply decolorizing agent (ethanol); this ONLY decolorizes gram-negative bacteria-washes crystal violet iodine complex out of gram-negative bacteria
  4. Apply secondary/counterstain to stain the decolorized bacteria red (safranin); if this step is left off, gram-negative bacteria would appear clear
  5. Gram positive bacteria have THICK peptidoglycan
  6. Crystal violet enters through the thick peptidoglycan cell wall of gram-positive bacteria
  7. Gram’s iodine reacts with crystal violet to form crystal violet iodine complex in the cytoplasm
  8. The cell wall is dehydrated by the alcohol while the crystal violet iodine stays inside of the cell
  9. Safranin ensures that contamination will be visible since it will result in seeing two colors purple-blue and pink-red

• Gram negative bacteria has a thin layer of peptidoglycan and an outer membrane layer made from lipopolysaccharides that can be dissolved by the decolorizing agent
• Grams iodine reacts with crystal violet to form the crystal violet iodine complex
• The decolorizing agent dissolves the outer lipopolysaccharide layer and washes the crystal violet out of the thin peptidoglycan layer
• The counterstain safranin stains the bacteria red since crystal violet has been washed out
• Older, dead bacterial cells may not retain crystal violet-iodine-complex (primary stain) because the cells are degraded
• Overheating during the heat fix causes the bacteria to burn and rupture the cell wall
• If the smear is too thick, the reagents may not reach all of the cells
• Leaving the dye on for too long may stain the glass slide which will make the bacteria not visible
• Picking up agar may make it hard to see bacteria

Streak plates and structural stains

• The streak plate method is a technique used to grow isolated colonies by streaking bacteria on agar plates; it dilutes the bacteria cells on the surface of the plate so they can grow in colonies; each colony should grow from a single bacterial cell, making them pure cultures
• The loop should be flamed in between streaks when inoculating a streak plate

Microscopy and stains true/false answers

• The needle is used to inoculate a broth. (False: it is used to inoculate agar deeps)
• The organism provided should have more than one type of growth visible on the agar slant. (False: agar slants are for pure cultures)
• Prokaryotes have membrane-bound organelles but their thick cell wall prohibits the observation (eukaryotes)
• Flagella are viewed with acid-fast staining (structural stains)
• In the hand-washing experiment, transient bacteria picked up by hands, normal flora that is naturally on our bodies would be removed in the hand washing section. The normal flora would most likely be found in the 5th (final) section.
• This experiment’s independent variable is handwashing time and the use of soap and the dependent variable colonies

Types of Bacterial Colony

• Different bacterial colony types are told apart by their form, margin, surface, color, size, color, margins (edges) and how translucent or opaque they are.

Controls

• In an experiment, control is used to to grow isolated colonies by streaking bacteria on agar plates to dilute bacteria cells on the surface of the plate so they can grow in colonies such that each colony grows from a single bacterial cell for pure cultures.

Gram Stains types

• Structural stains (special stains) are used to identify and study bacterial structures and observe bacterial endospores, capsules, and flagella and identify an unknown organism
• Endospores are called "resting bodies" and usually form in GRAM POSITIVE bacteria
  • They are dangerous when infected with endospore bacteria and form when nutrients or water are unavailable
  • Endospores do not metabolize or reproduce and are heat and chemical resistant
  • Endospores can survive harsh environmental conditions and remain dormant for a long time
  • Heat is applied because most endospores are resistant to stain
  • Endospores form inside the cell with endospore positions of free, subterminal, central, swollen, central, and terminal, swollen
• Capsules are a coat of uncharged polysaccharides secreted by bacteria around the cell in a round or oval shape and they are clear where only certain bacterial species have the genes to form capsule
• Capsule size is influenced by nutrients in the growth media and if bacteria are stained with a capsule they should be pinked with a clear halo
• Capsule virulence is disease causing capability
• Capsules prevent white blood cells from phagocytosis (digesting and killing of bacteria)
• Bacteria are not as resistant without capsules
• Motile bacteria move using flagella or move with gliding motion
• Motility can be determined with hanging drop, flagella stain, or semisolid agar deep with hanging drop (eukaryote)
• Flagella are thin and fragile and can be peritrichous- all around the bacteria, polar- at one or both ends of the cell, or are monotrichous, lophotrichous or amphitrichous

Answer questions

• Each flagella should only have one type like monotrichous, or peritrichous
• A mordant is used to coat the flagella forming a complex with crystal violet
• Forgetting iodine means crystal violet will wash away, and it would be impossible to determine if it is gram-positive or gram-negative
• The size is more accurate in a negative stain because negative staining doesn't require heat fixing so cells will not be distorted
• A darkfield technique gives a field similar in appearance to that seen in the negative stain
• Unlike nigrosin, acidic congro red does contain suspended particles but it can give the appearance of a negative stain because it has a negative charge and is repelled by the slight negative charge of most bacterial cells so the background gets stained instead of the bacteria
• Treponema denticola is easily missed, but a negative stain makes it visible by eliminating distortion with negative staining because no heat or distortion is used.
• Gram-negative cells stain red or pink.
• Gram-positive cells stain purple or blue.

Stain Colours

• After a structural stain, endospores stain green and the vegetative cells stain pink or red because the endospore retains malachite green in its thick walls and vegetative cells retain safranin counterstain
• Capsules stain purple with a clear halo against a dark background with the capsule itself appearing colourless after a structural stain
• Capsule structural stain is congo red
• Endospore structural stain is safranin
• Endospore position being Free, Central or Subterminal
• Most bacteria that possess flagella are rod shaped bacilli
• The cell doesn't appear green in the finished endospore stain because malachite green is water soluble and easily washes away during decolorization, but the thick walls of the endospore retain the malachite green, and the cell retains counterstain. (safranin)

Pathogenicity

• Capsules help cells evade phagocytosis allowing them to colonize faster than the body can eliminate
• Capsules are a protective barriers that prevents white blood cells from recognizing and engulfing the bacteria.
• Pathogenicity is aided by motility so bacteria can move toward ideal environments, attach to host cells, and swim to different tissues
• The advantage to clostridium is endospores which are highly resistant to harsh environments and survive even when nutrients and water are not available, remaining dormant for long periods of time and allows bacteria to survive longer and is thus harder to get rid of

Gram Staining and Flagella

• Crystal violet is used in both flagella and gram stains, but only gram-positive bacteria will retain crystal violet because of their different structure
• The mordant used in the gram stain is not used in the flagella stain
• Mycobacterium endospore stains green using malachite due the nature of dyes used
• A medium that is rich in polysaccharides increases the capsule size of bacteria enhancing polysaccharide containing capsule size
• Encapsulated streptococcus stains small, red, circular cells in chains with a clear halo against a dark background

pH

• pH is a scale to determine hydrogen ion concentrations to determine whether or not fluids are acidic, basic, or neutral and helps classify microbes based on their growth in different pH concentrations
• A tenfold change in hydrogen ion concentration changes for every unit of difference (multiply 10x per change in pH)
• If pH is too high or low this can affect solubility, denature proteins/enzymes, affect membrane transport and membrane potential
• Most bacteria are neutrophils, while fungi prefer acidic environment
• Archaea are found in all pH ranges
• All microbes keep their internal environment near neutral
• Most acidic pH is at 0, and the most basic is at 14
• Neutral pH reads 7
• Buffers stabilizes pH after adding it to growth media to prevent changes
• Fermentation of sugars relates to pH like when sugars convert into acids, this prevents other microbes from growing
• pH changes can identify bacteria using microbes that fall under alkaliphile, neutrophile, or acidophile by seeing which bacteria thrive in each environment'
• (5-2)/5x100=60% is uses to calculate the decrease
• (# of types - # of types) divided by # of types x 100 calculates percent increase
• Petroleum jelly creates a seal to prevent evaporation
• Microorganisms are hard to see in wet preparations because they can lack color and blend in with water
• Because they are long or because slides are too thick, immersion lens is unsuited in hanging drop
• Staining makes Protozoa is easier to see
• After completing observations under low power, high-dry objectives should be swung into position and requires only fine adjustment because the slide is mostly in focus and the focus should only need to use the fine focus as the working distance gets smaller as the lens gets closer.
• Increase light at higher magnifications using the illuminator or iris diaphragm which both affect the amount of light reaching the ocular lens
• Increased magnification decreases the field of vision
• Microscope objectives should be parfocal to easily switch between magnifications and focus on slides faster easily
• Oil immersion lens focuses closest to the slide when it is in focus
• Higher numerical aperture of lens and lower wavelength of light enhance resolving power
• Large fungal or algae organisms are easier to see with a low-power objective over the oil immersion objective
• Water would blur the image using immersion oil and the light will bend and distort

This lesson covers key concepts in microbiology related to bacterial staining techniques and the role of pH in microbial growth. Topics include Gram staining, capsule staining, pH scale and its effect on microbial life. Also covered is the impact of pH changes on microbial growth.