Micro Lab final

Human blood smear

Used to diagnose disease or conditions

ELISA is based on the principle that:

antibodies recognize and specifically bind to only one epitope.

ELISA

enzyme-linked immunosorbent assay

Agranulocytes

lymphocytes and monocytes

stage adjustment knobs

move stage sideways and back and forth so the slide can be underneath the `objective lens.

Stage

platform on which specimens are observed

MSAT plate will only have Gram:

positive bacteria

Eosinophils and and basophils

are involved in protecting the body against infections with large parasites. Basophils are also involved in allergic reactions.

A simple stain uses only one dye to determine: To do this, the procedures consists of 2 parts.

bacterial morphology: their shape and arrangement 1. 1. Prepare a bacterial smear 2. Stain the bacterial smear with a dye.

course focus knob

bigger of the two knobs, moves the stage up and down. Brings the specimen into focus. Used to bring objects into focus when using 4x or 10x objective lenses.

Differential media

contain ingredients that make different groups of bacteria appear different. They differentiate members of bacteria within the same group. An example: Blood Agar Plate (BAP), which allows the growth of many different bacteria, but distinguishes between their ability to degrade red blood cells.

selective medium

contain nutrients that allow certain bacteria to grow while inhibiting the growth of others. Examples: Mitis Salivarius Agar with Telluride (MSAT) and Maconkey Agar (MAC).

margin

entire (perfectly round), undulate, lobate, erose, and filimentous

Attached to the antibody is an enzyme, this is why is called an

enzyme-linked antibody. If the enzyme is attached to an antibody it will turn the substrate blue --> thus the antigen of interest is present.

There are three types of formed elements in blood:

erythrocytes (red blood cells), which carry oxygen and CO2, platelets, which are responsible for blood clotting and leukocytes (white blood cells), which are part of the body's immune system

Diagnosis of trichomoniasis

flagellated protozoan in between RBCs.

condenser lens

focuses light on specimen in one location. Small objects: adjusted all the way, so light is closest to the specimen.

Leukocytes are divided into

granulocytes (neutrophils, basophils, eosinophils) and agranulocytes (lymphocytes and monocytes).

PDA, potato dextrose agar

in lab will culture baker's yeast... microbe is one that is used daily to bake bread and to make beer or wine.

Basophils

involved in allergic reactions, inflammation release histamine

general-purpose medium

is a basic medium use to grow bacteria in quantities, and in order to observe colony morphology. They contain a carbon source such as glucose, a source of amino acids and nitrogen, water, and various salts needed for bacterial growth. Examples are Trypticase Soy Agar (TSA), used for growing and observing bacteria and Potato Dextrose Agar (PDA), for growing fungi.

Mitis salivarius agar (MSA)

is an enriched selective medium[1] we will use to isolate bacteria from the mouth.

Lympocytes

mainly involved in the adaptive immune response against a specific pathogen (NK cells are the exception: they are lymphocytes that are part of the innate immune response)

Stains do not work on mycoplasmas and mycobacteria b/c:

mycoplasmas do not have peptidoglycan mycobacteria that have a thick waxy layer of mycolic acid

Abundance of elements in the blood:

neutrophils make up 60-70% of the leukocytes lymphocytes 20-25% monocytes 3-8%, eosinophils 2-4%, and basophils 0.5-1%.

Granulocytes

neutrophils, eosinophils, basophils

Six colony characteristics are usually documented:

size, color, shape, edge, surface, and elevation Although, this is not enough for a definite identification of the bacteria. Also, the same bacteria will have different morphologies on different kinds of media.

Fine focus knob

the smaller of the knobs, sits on top of course knob. Used to move the stage up and down. Appropriate when using 40x to 100x objective lenses.

Monocytes

travel to sites of infection, and mature there into phagocytic macrophages. After phagocytosis, they travel to nearby lymph nodes, and form an important link between the innate and adaptive immune responses. Present to T cells.

Texture

(describes moisture) If the color is shiny= "moist" Flat = "dry"

blood elements model

1.

1. What is the purpose of the epsom salt in performing a fecal float? It is to: 2. Abundance of cells in the blood: 3. Which of these white blood cells travels to the site of infection or injury and matures into macrophages or dendritic cells?

1. Make the solution more dense than the parasite eggs, so the eggs float. 2. NLMEB 3. Monocytes

How to prepare a bacterial smear

1. bacteria are mixed with water on a glass slide and allowed to air dry so that they retain their shape. 2. The slide is then heat-fixed by a flame. Purpose: It kills the bacteria and attaches them to the glass slide so that they do not wash off as easily

Agar is a chemical derived from seaweed that is liquid when heated to 100°C but solidifies as it cools to 40°C. `` Once solidified, it remains solid even at higher temperatures. Agar ___ ___ provide significant nutrition for the microorganisms and remains solid as the bacteria start growing.

1. does not

Why must plates be incubated upside down?

All plates are kept inverted to prevent moisture from condensation on the lid to drop onto the surface of the plate during incubation. Moisture can change the consistency of the agar medium, which might affect the proper growth of microorganisms. It will also allow bacteria to spread more rapidly and thus hinder the isolation of colonies.

Neutrophils

A type of white blood cell that engulfs invading microbes and contributes to the nonspecific defenses of the body against disease.

colonies

Bacteria divide rapidly and form visible patches the surface of the agar called ___. - Contain billions of individual bacteria

The total magnification is the product of the magnification of the objective and ocular lense. 40x objective x 10 ocular =400

do not use oil immersion lens(100x) for fungal/protozoa slides.

What would happen if the decolorizer was left on for too long?

Crystal violet in the Gram-positive bacterial cell wall will dissolve and wash out.

Procedure for a fecal float:

Epsom salt will cause eggs to float. Wait 15 minutes. 100X magnification --> 400X

objective lens

First level of magnification- lenses closest to the observed specimen. 10x, 40x, 100x objective lenses. Occasionally, 4x.

elevation

Flat, raised, convexed, pulvinate, and crateriform.

Signs of malaria

High fever/ chills, anemia, jaundice and dark urine= "black water fever" Diagnosed from blood smear

stage clips

Holds the slide in place

Once the bacteria are stained, for the bacterial smear, you can view them with __x ocular lens and ___x objective. Then work your way up to the highest magnification objective lense ___ x increasing the light as necessary. Lower magnifications will not provide enough detail for you to determine bacterial morphology

Largest magnification ocular lens (25x) and start by observing the slide with the smallest objective (4×) Highest magnification objective lens (40x)

MSAT, mitis salivarius agar with telluride

Medium used to isolate bacteria from your mouth cavity. Only benign bacteria will grow on this medium. You can transfer bacteria from this medium to other media for study. Petri dishes with MSAT are easily recognized by the dark blue color of the medium.

Dedritic cells

Monocytes can mature into this cell. Wait in tissues. Phagocytic travel to lymphnode and present peices of antigen to MHC complexes. Activates addaptive immune.

Blood borne parasite: Malaria

Plasmodium flaciparum --> transmitted by female Anopheles mosquito. Parasite travels to the liver --> invade RBCs. 2-3 days, Plasmodium bursts from cells.

How to do a gram (differential) stain:

Prepare a bacterial smear and heat-fix it. Place the slide on the slide holder. Flood the smear with the primary stain (crystal violet), leave it on for one minute, then rinse with water. Flood the smear with mordant (Gram's iodine), leave it on for one minute, then rinse with water. Flood the smear with decolorizer (isopropanol or ethanol), leave it on for 10-15 seconds, then rinse with water. Flood the smear with the counterstain (safranin), leave it on for one minute, then rinse with water. Blot the slide dry with bibulous paper. Observe the stained slide under the microscope at high magnification (1000x). Report your Gram-stain results (Gram-positive or Gram-negative), shape, arrangement, and magnification

Shape (form)

Punctiform, circular, filimentous, irregular, rhizoid, spindle

Diaphragm

Regulates the amount of light on the specimen. Open or smaller objects slightly closed for larger objects

6 characteristics to describe bacterial morphology.

Size, color, texture, shape(form), elevation and margin

epitope

Small, accessible portion of an antigen that can be recognized. Usually a protein or carbohydrate.

At the end of a differential stain, why are gram + and gram - different colors?

The difference in the structure of their cell walls. Gram-positive bacteria have a thick layer of peptidoglycan outside their cell membrane, while Gram-negative bacteria have a thin layer of peptidoglycan and an additional outer membrane.

Trypanosomiasis (African Sleeping Sickness)

Trypanosoma brucei transmitted: Tsetse fly fever, inflammation of the meninges and the brain. Headache, neurological signs/symptoms, drowsiness. Fatal if not treated.

Trypanosomiasis (changas disease)

Trypanosoma cruzi Transmission: Kissing bug can live in macrophages/ erythrocytes. Fever, swollen lymph nodes, enlarged spleen Inflammation of heart muscle --> CHF

TSA, trypticase soy agar

This is a general-purpose medium that you will use for activities involving bacteria after they have been cultured on MSA. Petri dishes containing this medium, as well as tubes containing the liquid version of this medium (without agar) will be marked with a bright pink line

Maconkey Agar (MAC)

allows the growth of most Gram-negative bacteria, but most Gram-positive bacteria cannot grow on it. MAC will be explained in more detail in a later handout.

Inoculation

is the intentional introduction of microorganisms onto a sterile growth medium (usually sterilized in an autoclave)

Enriched medium

is usually a basic medium enriched with a variety of ingredients that enable a wide range of organisms to grow, especially the most fastidious[1] ones.

Ocular

provide 2nd magnification. Observe specimens by looking through the ocular lenses

agar is dissolved, the bottle is placed in an autoclave where it is heated at high pressure (121°C, 15 psi). This kills all organisms that are in the medium so that it becomes ___.

sterile