Handwashing and Microbiota

Lab 1: Handwashing

• Normal microbiota refers to the microorganisms that are normally and consistently found in or on the body in a healthy person.
• Transient microbiota refers to microorganisms that are temporarily present on or in the body.
• Skin microbiome refers to the collective community of microorganisms residing on the skin.
• Ignaz Semmelweis was an early pioneer of antiseptic procedures, advocating for handwashing to prevent the spread of disease in the 1800s.
• According to the CDC, hand hygiene is the single most important procedure for preventing healthcare-associated infections.
• Healthcare providers wash hands less than 50% of when needed.
• Soap helps in the removal of microorganisms and dirt by emulsifying fats and reducing surface tension.
• Scrubbing physically removes microorganisms from the skin.
• Components of the skin's natural flora and oils prevent handwashing from removing all bacteria.
• Hands are washed in a healthcare setting to prevent the spread of infections between patients and healthcare workers.
• Handwashing typically removes transient microbiota.
• Surgeons and nurses scrub thoroughly before surgery to remove as many microbes as possible, including normal microbiota, to prevent infections.
• Sterilizing a surgeon's skin is not possible due to the presence of normal microbiota and skin structure, but the number of microbes is reduced.
• Ignaz Semmelweis first demonstrated the importance of handwashing in Europe during the 1800s; he discovered the link between hand hygiene and reduced mortality rates in maternity wards.

Lab 2: Microscopy

• A brightfield compound microscope is a type of microscope that uses visible light to illuminate a sample, producing a bright background against which the sample can be seen.
• The stage is the platform where the specimen to be viewed is placed.
• The arm is the part of the microscope that connects the base to the head and is used to carry the microscope.
• The body tube is the part of the microscope that holds the objective lenses and the ocular lens, ensuring proper alignment of the optical components.
• The condenser focuses light onto the specimen.
• The iris diaphragm controls the amount of light that reaches the specimen.
• Objective lenses are the primary lenses on a microscope that magnify the image of the specimen.
• The ocular/eyepiece lens (10x) further magnifies the image produced by the objective lens.
• Coarse adjustment is used for initial focusing of the specimen at lower magnifications.
• Fine adjustment is used for precise focusing after using the coarse adjustment.
• Field of vision is the area visible through the microscope's eyepiece.
• Depth of field refers to the thickness of the specimen that is in focus at one time.
• Magnification is the process of enlarging the apparent size of an object.
• Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.
• Scanning lens (4x) total magnification is 40x (4x objective lens * 10x ocular lens).
• Low power objective (10x) total magnification is 100x (10x objective lens * 10x ocular lens).
• High power objective (40x) total magnification is 400x (40x objective lens * 10x ocular lens).
• Oil immersion lens (100x) total magnification is 1000x (100x objective lens * 10x ocular lens).
• Resolution/resolving power is the ability of a microscope to distinguish two objects as separate entities.
• Refractive index is a measure of how much light bends when it passes from one medium to another.
• Focal point is the point at which light rays converge after passing through a lens.
• Parfocal means that once a specimen is in focus with one objective lens, it will remain nearly in focus when switching to another objective lens.
• Working distance is the distance between the objective lens and the specimen when the specimen is in focus.
• Brightfield microscopy uses white light.
• Microbiome refers to the collective community of microorganisms in a particular environment.
• The parts of the microscope include the base, arm, stage, body tube, coarse and fine focus knobs, objective lenses, ocular lens, condenser, and iris diaphragm.
• Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.
• The microscope is used by first placing the specimen on the stage, focusing with the coarse adjustment knob, and then using the fine adjustment knob for sharper focus at each magnification.
• Oil is used with the oil immersion lens to improve resolution by reducing light refraction
• Resolving power can be increased by using light with a shorter wavelength or by using a lens with a higher numerical aperture.
• As magnification increases, the field of view decreases.
• Controls on a microscope that affect the amount of light reaching the ocular lens include the iris diaphragm and the light intensity adjustment knob.
• Wet mount technique involves placing a specimen in a drop of liquid on a slide and covering it with a coverslip.
• Depression slide technique involves placing a specimen in a special slide with a concave depression to observe motility and prevent crushing of the sample.
• Motility is difficult to determine in wet mount slides due to the limited space and rapid drying.
• Advantages of the depression slide include allowing more space for motility and preventing the crushing of delicate specimens.
• The oil immersion lens (100x) is necessary to observe the morphology of prokaryotes due to their small size.
• Prokaryotes can be differentiated from eukaryotes using a microscope by observing the presence or absence of a nucleus and other membrane-bound organelles.
• Relative sizes of microorganisms: Bacteria and archaea are generally smaller than fungi, protozoa, and algae.
• Basic shapes (morphology) of bacteria: bacillus (rod-shaped), coccus (spherical), and spiral.

Lab 3: Environment and Aseptic Technique

• Microbiome refers to the collective community of microorganisms in a particular environment.
• Chemically defined medium is a growth medium composed of precisely known chemical components.
• Complex media are growth media that contain ingredients of unknown chemical composition.
• Meat extracts and peptones provide nutrients, vitamins, and growth factors.
• Nutrient broth is a liquid medium containing nutrients for bacterial growth.
• Nutrient agar is a solid medium containing nutrients and agar as a solidifying agent for bacterial growth.
• Agar is a polysaccharide derived from seaweed, used as a solidifying agent in microbiological media.
• Autoclave/steam sterilization/autoclaving is a method of sterilization using high-pressure steam.
• Petri plates are shallow, transparent dishes used to culture microorganisms on solid media.
• Inoculation is the process of introducing microorganisms into a culture medium.
• Incubation period is the time during which a culture is allowed to grow.
• Turbid describes a broth culture that is cloudy due to microbial growth.
• Colony is a visible population of microorganisms growing on a solid medium.
• Colony-forming unit (CFU) is a measure of viable microbial cells that can form a colony on an agar plate.
• Flocculent describes a broth culture with suspended clumps or aggregates of microorganisms.
• Pellicle describes a surface membrane or film formed by microorganisms at the air-liquid interface of a broth culture.
• Sediment is a deposit or accumulation of microorganisms at the bottom of a broth culture.
• Agar is used in the preparation of some forms of growth media because it is a solidifying agent that does not melt at sterilization temperatures and is not degraded by most microorganisms.
• Petri plates are incubated inverted to prevent condensation from dripping onto the agar surface and contaminating the culture.
• Condensation on the agar is undesirable because it can spread microbial growth and make it difficult to isolate individual colonies.
• Turbid, pellicle, flocculent, and sediment are terms used to describe growth in a broth culture.
• Whole colony appearance (circular, irregular, rhizoid), margin (entire, lobate, undulate, serrate, filamentous), and elevation (flat, raised, convex, umbonate) are terms used to describe colony appearance.
• Contaminant is an unwanted microorganism in a culture.
• Aseptic means free from disease-causing microorganisms.
• Aseptic technique is a set of procedures used to prevent contamination by microorganisms.
• Sterilize means to make something free from all living microorganisms.
• Broth culture is a liquid medium used for growing microorganisms.
• Agar deep/semisolid agar deep refers to agar media in tubes that is either solid or semi-solid.
• Inoculating loop is a tool used to transfer microorganisms from one medium to another.
• Inoculating needle is a straight wire used for transferring microorganisms, especially for stab cultures.
• Bacterial motility can be determined using a motility test medium, flagella stain, or by observing growth patterns in semisolid agar.
• Primary uses of slants are for maintaining stock cultures, deeps are for testing the oxygen requirements of microbes, and broths are for obtaining a large number of cells.
• For a bacterium, forming a pellicle provides access to oxygen and nutrients at the surface of the broth.
• Proper aseptic technique includes sterilizing equipment, working near a flame, and minimizing exposure of sterile materials to the air.
• 15 psi must be applied to steam in order to raise the temperature of the steam to 121°C for 15-20 minutes and destroy spores of spore-forming bacterial species, typically done using an autoclave.

Lab 4: Isolation and Cultivation of Bacteria

• Plates are incubated upside down to prevent condensation from dripping onto the agar surface.
• The value of an autoclave is its ability to sterilize materials by using high-pressure steam.
• Three methods commonly used for isolating bacteria are the streak plate method, the pour plate method, and the spread plate method.
• Isolating a single type of bacteria is useful for studying its characteristics and performing specific tests.
• Quantifying amounts of bacteria is useful for determining the concentration of bacteria in a sample and monitoring growth.
• Types of samples that could be assayed for the number of bacteria present include water, soil, food, and clinical specimens.
• Culture plates with fewer bacteria are likely grown in a high salt concentrate like MSA
• Given a dilution series setup, dilutions can be determined by multiplying the dilution factors at each step.
• CFU/ml can be calculated by dividing the number of colonies by the volume plated and multiplying by the dilution factor.
• TNTC stands for "Too Numerous To Count," meaning there are too many colonies to accurately count.
• TFTC stands for "Too Few To Count," i.e., <25 CFU.
• A countable plate typically has between 30 and 300 colonies.
• Problems with streak plate technique can be identified by uneven distribution of colonies or contamination.
• Contamination is the presence of unwanted microorganisms in a culture.
• Selective media are used to inhibit the growth of certain microorganisms while allowing others to grow.
• Differential media are used to distinguish between different types of microorganisms based on their biochemical reactions.
• Enrichment media are used to enhance the growth of specific microorganisms by providing specific nutrients.
• Mannitol salt agar (MSA) is classified as both selective and differential, contains mannitol, salt, and a pH indicator, and is used to differentiate bacteria based on mannitol fermentation.
• Bacterial groups that grow well on MSA are Staphylococcus species because they tolerate high salt concentrations.
• Eosin-methylene blue (EMB) agar is classified as both selective and differential, contains eosin and methylene blue dyes, and is used to differentiate bacteria based on lactose fermentation.
• Species that exhibits a metallic green appearance on EMB agar is E. coli.
• Species that acidifies and turns MSA media a yellow color are those that ferment mannitol, like Staphylococcus aureus.

Lab 5: Simple Staining and Gram Staining

• Visualization of living microorganisms is difficult because they are often transparent and motile.
• A smear is a thin film of microorganisms spread on a slide for staining.
• Heat-fixation accomplishes adherence of the microorganism to the slide, kills the bacteria, and coagulates proteins.
• A stain (dye) is a substance used to color microorganisms for better visibility under a microscope.
• A chromophore is the colored part of a stain.
• Basic stains interact with cells by binding to negatively charged components in bacterial cells.
• An acidic stain is a stain with a negatively charged chromophore that stains alkaline structures.
• A simple stain uses a single dye to provide contrast, making it easier to observe cell shape and arrangement.
• A direct stain stains the microorganism.
• A negative stain stains the background, making it useful for observing cell shape and capsules.
• A differential stain uses multiple dyes to distinguish between different groups of bacteria, used to identify microorganisms.
• Gram stain is a differential stain used to classify bacteria based on cell wall structure and is based on differences in peptidoglycan layer thickness.
• A primary stain is the first stain applied in a differential staining procedure, like crystal violet in Gram staining.
• A mordant is a substance that helps a stain bind to a target molecule, such as Gram's iodine.
• A decolorizing agent removes the primary stain from certain cells, such as ethyl alcohol in Gram staining.
• A secondary stain, or counterstain, is applied after decolorization to stain cells that lost the primary stain, such as safranin in Gram staining.
• The purpose of heat fixation is to adhere the microorganisms to the slide, kill the bacteria, and coagulate proteins.
• Heat fixation is performed by passing the slide quickly through a flame several times.
• If a smear is too thick, visualization may be difficult due to clumping and reduced light penetration.
• If a smear is too thin, there may not be enough cells to observe.
• The purpose and order of reagents in Gram staining:
  • Crystal violet/primary stain stains all cells purple.
  • Gram's iodine/mordant fixes the crystal violet to the cell wall.
  • Ethyl alcohol/decolorizing agent removes the crystal violet from Gram-negative cells.
  • Safranin/counterstain stains Gram-negative cells pink.
• Inaccurate Gram stain results can occur due to old cultures, improper technique, or thick smears.
• The mordant step in the Gram stain could be omitted.
• Gram staining is useful in the clinical lab for rapid identification of bacterial infections.
• Gram-staining information can influence the choice of antibiotic by indicating whether the bacteria are Gram-positive or Gram-negative, which affects antibiotic susceptibility.