Microbiology Lab Midterm Study Guide

Check your Understanding:

How many nanometers is 10 μmeter? - 1000 nm

Protein Catabolism

- Catabolism = Decomposition (break down) of complex organic molecules to smaller molecules which release energy. - Proteins: Large, organic macromolecules. Polymers, the monomers are amino acids Small chains= peptides, larger molecules =polypeptides Covalent bond = peptide bond Elements: C, H, O, N, and [ sulfur, S]

Distinguish chemically defined and complex media.

- Chemically defined media: exact chemical composition is known - Complex media: extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch

Gram Stain

- Classifies bacteria into gram-positive or gram-negative - Gram-positive bacteria have thick peptidoglycan cell walls-stain purple - Gram-negative bacteria have thin peptidoglycan cell walls and a layer of our phospholipid bilayer with lipopolysaccharides-stain pink

MRVP Test

- A differential medium - A glucose supplemented broth - It distinguishes organisms that produce a large amount of acid by glucose fermentation from organisms that produce acetoin (a neutral compound) as a by-product of glucose catabolism - MRVP medium is used for: Methylene red (MR) test & Voges-Proskauer(V-P) test

Acid-Fast Staining

- A differential stain - Acid-fast microorganisms do not decolorize with an acid-alcohol wash - The mycolic acid (lipid) in the cell wall makes the cell wall almost impermeable - The primary dye is mixed with phenol and the smear is heated to aid penetration of the primary dye - The primary stain -Carbolfuchsin -is retained by the cells after alcohol wash (color=pink to red) - Any other cells in the smear will be colored by the secondary dye, Methylene Blue (color=Blue)

Broth Media

- A liquid medium used for growing cells

Proper Attire Includes:

- A shirt that covers torso and arms - Long Pants - Short Hair or long hair thats tied back

Neutrophils

- A type of white blood cell that engulfs invading microbes and contributes to the nonspecific defenses of the body against disease. - They kill and digest bacteria and fungi. They are the most numerous type of white blood cells and your first line of defense when infection strikes.

Lymphocytes

- A type of white blood cell that makes antibodies to fight off infections - They create antibodies to fight against bacteria, viruses, and other potentially harmful invaders.

Monocytes

- A type of white blood cell that transforms into macrophages, extends pseudopods and engulfs huge numbers of microbes over a long period of time - They have a longer lifespan than many white blood cells and help to break down bacteria.

Eosinophils

- A white blood cell containing granules that are readily stained by eosin. - They attack and kill parasites and cancer cells, and help with allergic responses.

Fixing will:

- Adhere sample to the glass slide - Kills the microorganism - Preserves the sample

Differential media

- Allow distinguishing of colonies of different microbes on the same plate - Some media have both selective and differential characteristics

Agar Slants

- Are test tubes containing solid culture media that were left at an angle while the agar solidified; are easier to store and transport than Petri plates

Acid-Fast Stain

- Binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alcohol - Used for the identification of: Mycobacterium & Nocardia

Negative Staining for Capsules

- Capsules are a gelatinous covering that do not accept most dyes - Suspension of India ink or nigrosin contrasts the background with the capsule, which appears as a halo around the cell

Define the following terms: Carbohydrate, Catabolism, and hydrolytic enzymes

- Catabolism = Decomposition (break down) of complex organic molecules to smaller molecules that release energy. - Carbohydrate = Organic macromolecules that have the general formula (CH2O) n Carbohydrates based on size: Monosaccharide Oligosaccharides Polysaccharides Carbohydrates are the substrate for hydrolytic reactions in bacteria cells. - Hydrolytic enzymes which can be exo-enzymes breakdown carbohydrates by the addition of water molecules.

Agar

- Complex polysaccharide - Used as a solidifying agent for culture media in Petri plates, slants, and deeps - Generally not metabolized by microbes - Liquefies at 100 oC - Solidifies @ ~40 oC

Types of Light Microscopy

- Compound light microscopy (stained bacterial smear) - Dark-field microscopy (unstained bacterial cells; the cells are small and no detail is needed) - Phase-contract microscopy (unstained live tissue when it is desirable to see some intracellular detail) - Differential interference contrast (DIC) microscopy (unstained live cells in which intracellular structures are shown in color) - Fluorescence microscopy (a sample that emits light when illuminated with ultraviolet light) - Confocal microscopy

Check Your Understanding: Through what lenses does light pass in a compound microscope?

- Condenser lenses - Objective lenses - Ocular lens

Direct Measurement of Microbial Growth Plate Counts

- Count colonies on plates that have 30 to 300 colonies (CFUs) - To ensure the right number of colonies, the original inoculum must be diluted via serial dilution - Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method)

Isolation & Inspection

- Culture = the observable growth that happens in or on medium - Isolation : Serial Dilution & streak plate to prepare pure culture. (Individual bacterial cells are separated, and grow in discrete mound of cells called Colony) - Colony = groups of cells large enough to be seen without a microscope, all from one cell or CFU - Inspection: Staining of the bacteria, biochemical testing, etc.

Enrichment Culture

- Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels. - Usually a liquid

Endospore Staining

- Endospores are resistant, dormant structures inside some cells that cannot be stained by ordinary methods - Primary stain: malachite green, usually with heat - Decolorize cells: water - Counterstain: safranin - Spores appear green within red or pink cells

Safety Equipment Includes:

- First Aid Kit (Minor cuts & Scrapes) - Fire Extinguisher (Small fires & Not to be used on people) - Eyewash (Chemical contact with face & eyes.) - Fire Blanket (Smother's fires especially on people) - Safety Shower (Use in case chemical comes into contact with skin) - Safety Data Sheet (SDS should be used before working with chemical spills or other accidents.) - Phone (In case of emergency call for fire or police or other emergency contacts.) - Evacuation Route (Use the evacuation route to flee the emergency) - Lab Partner: Never work alone.

Flagella Staining

- Flagella are structures of locomotion -Motility - - Uses a mordant and carbolfuchsin to thicken appearance of flagella, making them visible under the light microscope

Personal Protective Equipment Purpose & Includes:

- Helps protect vital areas of the skin and face from harm. - Lab Coat - Safety Glasses - Latex or Nitrile Gloves

Diagram the path of light through a compound microscope (L.O)

- Illuminator - Condenser - Through the specimen - Objective lenses - Ocular lens

Commonly used dyes for simple stains

- Methylene Blue - Carbolfuchsin - Crystal Violet - Safranin

Simple Stains

- Methylene Blue - Safranin

List the units used to measure microorganisms (L.O)

- Microorganisms are measured in micrometers μm and nanometers(nm)

Learning Objectives: List different types of White Blood Cells

- Monocytes. - Lymphocytes. - Neutrophils. - Basophils. - Eosinophils.

Recognize different arrangements of flagella

- Monotrichous - Lophotrichous - Amphitrichous - Peritrichous - Axial filaments

What should we use if normal Staining do not Work?

- Negative Staining: Does not stain the microorganisms but stains the background. - Appearance: the bacteria is clear against a stained background - Negative Stain: Do not fix the slide! No rinsing! Use special dyes which are negatively charged and too big to enter the cells Ex: Nigrosin & Eosin

Commonly used dyes for negative stain:

- Negrosin - Eosin

Safe Environment:

- No food or drinks in lab - Clean up after yourself - Put equipment away & throughly wash out glassware - Keep space around safety equipment clear & easily accessible

How do exo-enzymes help in identification of bacteria?

- Not all bacteria have the same set of exo-enzymes. - The presence of an exo-enzyme is determined by looking for changes in the substrate outside of a bacterial colony.

Microorganisms are fixed(attached) to the slide, which kills the microorganisms

- Pass the slide over the flame 2X - Place on 60oC slide warmer for 10 minutes - Hold at 1cm above an electric incinerator for 20 seconds or - Cover with 95% methanol for 1 minute, let it drip and air dry

Basic Rules of Lab Safety:

- Proper Attire - Personal Protective Equipment (PPE) - Safe Environment - Safety Equipment - Following Directions

Starch Agar test:

- Starch agar medium = basic nutrients + Starch - A differential medium - Iodine is added after incubation - Dark blue or black area-starch is present - Clear area -No starch b/c bacteria used its exo-enzyme (ex: amylase) to degrade starch! - Thus: Bacteria with clear area has the exo-enzyme amylase or oligo-1,6-glucosidase

Selective and Differential Media

- Suppress unwanted microbes and encourage desired microbes - Contain inhibitors to suppress growth

Urea Agar test:

- Tests for the presence of Urease enzyme - Contains: Peptone Glucose Urea Phenol Red - indicator At pH 6.8 -Yellow At alkaline pH (8.4 and above)-Hot pink8

Compound light microscopy

- The image from the objective lens is magnified again by the ocular lens. - Resolutions the ability of the lenses to distinguish two points - A microscope with a resolving power of 0.4 nm can distinguish between two points at least 0.4 nm apart - Shorter wavelengths of light provide greater resolution - The Refractive Index is a measure of the light-bending ability of a medium - Light may refract after passing through a specimen to an extent that it does not pass through the objective lens - Immersion oil is used to keep light from refracting - Brightfield illumination (Dark objects are visible against a bright background & Light reflected off the specimen does not enter the objective lens)

Learning Objectives: Distinguish between "Sign" and " Symptom"

- The sign is objective evidence of disease - A symptom is subjective. "Sign" and "Symptom" are both medical terms with different medical meanings. While symptoms are problems that a patient notices or feels, signs are whatever a physician can objectively detect or measure. For example, if a patient feels hot, this is a symptom.

Basophils

- These small cells seem to sound an alarm when infectious agents invade your blood. They secrete chemicals such as histamine, a marker of allergic disease, that help control the body's immune response.

Inoculation & Incubation

- To cultivate, or culture, microorganisms: Aseptically introduce a tiny sample , inoculum, into a container of sterile nutrient medium. - Aseptic = Refers to prevention of infection. - All instruments must initially be sterile. - Sterile = Complete absence of viable microbes. - Incubation: Inoculated media is placed in a temperature-controlled chamber, or incubator.

Simple Stain

- Use of a single basic dye - Highlights the entire microorganism to visualize cell shapes and structures

Agar Deep

- When agar is allowed to solidify in the bottom of a test tube; used to grow bacteria that prefer less oxygen than is present on the surface of the medium

Culturing Microorganisms: The five I's

1. Inoculation 2. Incubation 3. Isolation 4. Inspection 5. Identification

Anemia

A condition in which the blood is deficient in red blood cells, in hemoglobin, or in total volume.

Malaria

A disease caused by mosquitoes implanting parasites in the blood.

Hereditary disease

A disease in which genetic factors have been transmitted from parent to offspring

Congenital disease

A disease that begins before birth and is evident at the time of birth

Sickle Cell Anemia

A genetic disorder that causes abnormal hemoglobin, resulting in some red blood cells assuming an abnormal sickle shape

Learning Objectives: Streak Plate Method of Isolation

A method of isolating a culture by spreading microorganisms over the surface of a solid culture medium

Kinyoun Stain

A modified version of the Ziehl Neelsen stain (Acid-fast) which changes the concentration of the dye & the phenol thus there is no heat needed

Culture Medium

A nutrients prepared for microbial growth - usually sterilized before inoculated

Learning Objectives: Understand the use of stained blood smear in clinical laboratory

A procedure in which a sample of blood is viewed under a microscope to count different circulating blood cells (red blood cells, white blood cells, platelets, etc.) and see whether the cells look normal.

Microscopy: The Instruments

A simple microscope consists of one lens; a compound microscope has multiple lenses.

Smear

A thin film of a material containing microorganisms spread over a slide

Learning Objectives: Stained Blood Smear Slide

A thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various blood cells to be examined microscopically.

Learning Objectives:The concepts behind properly prepare slides for microbiological examination, including performing wet-mount and/or hanging-drop preparations.

Advantages of Hanging drop: - Not flattened, so they behave more normally - More details - Can't get out of the bubble, so they don't leave field of view - Stays moist Disadvantages of wet mount: - Kept having to focus in and out - Things move in and out of view - They dry out due to light - Behaviors may change due to the spotlight.

Learning Objective: Describe why agar is used in culture media.

Agar: - Is Complex polysaccharide - Used as solidifying agent for culture media in Petri plates, slants, and deeps. - Generally not metabolized by microbes ; Liquefies at 100°C / Solidifies at ~40°C

Nucleoli

Areas in nucleus with high concentrations of protein and RNA molecules; ribosomes assembled here

Week 2:

Aseptic Technique

Check Your Understanding: What does it mean when a microscope has a resolution of 0.2 nanometer?

Can distinguish objects greater than or equal to 0.2nm

Protein Hydrolysis

Bacteria can hydrolyze peptide: - Peptide hydrolysis release amino acids, which are used as a source of Carbon and energy - Bacteria use peptide as source of Carbon & energy when there are no Carbohydrate available! - However, the major function of amino acids is their role in anabolic reactions

How are blood smears used to diagnose diseases?

Blood cells can be studied by spreading a drop of blood thinly on a glass slide, drying it, then staining the cells. Preparations obtained in this way are called stained blood smears. Examination of a peripheral blood smear can help in the diagnosis of communicable and non-communicable diseases that affect red and white blood cells.

Leukemia

Blood condition of white cells; malignant (cancerous) condition.

Identify the function of Capsules, flagella, and the endospores

Capsules: - Many bacteria secrete chemicals that adhere to their surfaces, forming a viscous coat. This structure is called a capsule when it is round or oval in shape, and a slime layer when it is irregularly shaped and loosely bound to the bacterium. Flagella: - The most common means of motility, are thin proteinaceous structures that originate in the cytoplasm and project out from the cell wall. They are very fragile and are not visible with a light microscope. Endospores: - Are formed by several genera in the orders Bacillus and Clostridia's.* Bacillus and Clostridium are the most familiar genera. Endospores are called "resting bodies" because they do not metabolize and are resistant to heating, various chemicals, and many harsh environmental conditions

Staining

Coloring microorganisms with a dye that emphasizes certain structures

Pure Culture

Contains only one species or strain

Culture Media

Culture Medium: - Material prepared in a laboratory for the growth of microorganisms = Culture Media - Minimal medium culture: Source of Carbon, Source of Nitrogen, Source of phosphorous & sulfur, Source of energy, Minerals, Growth factors

Hematopathology

Diagnosis blood disease And blood forming organs

Analyze bacteria growth on MSA, EMB and MAC plates.

EMB: - Black colonies with a green sheen => Gram negative, Lactose fermentor - Colorless colonies => Gram negative; and non-lactose fermentor MSA: - High Salts: Prevents growth of non-halophiles - Mannitol: Organisms that can ferment will grow yellow colonies - PH Indicator: Fermentation of Mannitol turn the medium acidic => Yellow A Differential Medium-Mannitol Salt AgarIt can be also considered a selective medium

Week 6

Exercise 13-Carbohydrate Catabolism & Exercise 14-Fermentation

Week 3:

Exercise 2 & 3; Wet Mount & Hang Drop

Week 4

Exercise 5; Smear and Simple Stain

Week 5

Exercise 8; Acid-Fast Stain & Exercise 9; Structural stains

Is there a test to identify other carbohydrates used by a Fermentative Bacteria?

Fermentation Tubes: -a differential medium- - Is used to determine production of acids and gas from carbohydrates - Fermentation tube medium = basic nutrients + Peptone Desired carbohydrate at 0.5-1% concentration Inverted glass tube (Durham) to capture released gas Acid-base indicator (phenol red) Yellow in color: Fermentation that produces acid Red in color: neutral (or alkaline due to long incubations)

End products of Carbohydrate Fermentation

Fermentation of Carbohydrates (Sucrose, lactose, fructose, glucose, etc.) - Produce different by-products - Acidic compounds or Neutral compounds - Production of the by-products depend on: Type of microorganism Substrate Incubation time - MRVP test is used to answer which by-product is produced

Staining techniques (Gram Stain)

Fix the slide 1. Apply primary stain 2. Apply mordant 3. Apply decolorizing agent 4. Apply secondary stain, or counterstain

Gelatin Hydrolysis Test

Gelatin Hydrolysis test: - Gelatin - Large protein molecule - Melts at 50oC - Solidifies at below 25oC - Bacteria exo-enzyme gelatinase hydrolyzes Gelatin - Thus in the presence of gelatinase it does not solidify at low temp.

Mononucleosis

Infectious disease marked by increased numbers of mononuclear leukocytes and enlarged cervical lymph nodes

Learning Objectives: Why do we use Aseptic technique in Microbiology Labs

Inoculation: - Introduction of microorganisms (bacteria) to a culture medium without introducing unwanted microorganisms or contamination. - Nutrient medium (Broth, Agar slant, Agar deep, agar plate [petri dish] - Nutrient media are sterilized - Sterilization: autoclaved so it is free of all life - Purpose: to grow a specific microorganism - Aseptic technique: Procedure used in the lab to exclude contaminants

Inoculum

Introduction of microbes into medium using aseptic technique

Week One: Lab Safety

Introduction to Lab Safety

Colony

Is often called a colony-forming unit (C F U)

Pernicious Anemia

Lack of mature erythrocytes caused by inability to absorb vitamin B12 into the bloodstream

Litmus milk Test

Litmus milk test: - Casein = a protein in milk - Litmus milk test: - Skim milk - Litmus -an indicator; blue in color - Tests for peptonization (hydrolysis of Casein protein) - Tests also for Lactose fermentation - Tests for Litmus reduction - Tests for Curd formation

Acute Lymphocytic Leukemia

Malignant, immature lymphocytes multiply in the blood, bone marrow, and lymphatic system.

Mordant

May be used to hold the stain or coat the specimen to enlarge it

Streak Plate

Method is used to isolate pure cultures

Culture

Microbes growing in or on a culture medium

Sterile

No living microbes

Understand the concepts behind starch hydrolysis & OF tests, and correctly interpret these tests results

OF medium test: - OF medium = basic nutrients in a semisolid deep + - A specific carbohydrate ( Glucose) @ high concentration + - Peptone at low concentration + - pH indicator, Bromophenol Blue: - turns yellow in acidic environment -Carbohydrate catabolism -fermentative, but can also be oxidative - turns dark blue in alkaline environment - (peptone catabolism)

Week One: Microscopy

Observing Microorganisms under a Microscope

Differentiate oxidative from fermentative catabolism

Oxidative: - Glucose = a mono saccharide = carbohydrate - Glucose ---> CO2+ H2O - A catabolic reaction - Oxidative reaction - Requires O2 - Endo-enzyme catalyzes the reaction Fermentative: - Glucose = a mono saccharide = carbohydrate - Glucose ---> small organic molecules (acidic) - A catabolic reaction - Fermentation - Does not require O2 - Endo-enzymes catalyze the reactions

Following Instructions

Probably the most critical piece of safety advice when working in a lab environment is to carefully follow all of the rules and procedures. That includes the ones we've already covered. Following instructions promotes a safe and productive lab environment.Even something as simple as mixing two chemicals such as acid and water can be dangerous if you don't follow instructions. In that case, SDS or Safety Data Sheets provide critical information and instructions when working in the lab with chemicals and other substances.

Negative Staining

Staining the background instead of the cell

Stains:

Stains consist of a positive and negative ion, one of which is colored (chromophore)

Basic Dye

The chromophore is a cation

Acidic Dye

The chromophore is an anion

Define total magnification and resolution (L.O)

Total Magnification - Objective lens x Ocular lens Resolution - The ability of the lenses to distinguish fine detail and structure between two points

Learning Objective: Distinguish true motility from Brownian movement.

True Motility: - True motility is movement of the cell by appendages. - The cells move to and from. - The movement is more obvious. Brownian Movement: - Brownian motion is the bombardment of cells by water molecules. - The cells appear to be vibrating.

Learning Objective: Provide the rationale for Aseptic Technique

Used in microbiology to exclude contaminants, unwanted microbes

Special Stains

Used to distinguish parts of microorganisms - Capsule stain - Endospore stain - Flagella stain

Learning Objectives: Understand the value of patients signs and symptomsfor correct disease diagnosis

When a client is experiencing signs and symptoms of disease, the disease is considered to be in the clinical phase of its clinical course.

What do you look for in a properly stained Slide?

When examining a properly stained slide, the examiner should note the following characteristics: - the presence of a single type or several types of organisms - the predominant type of organism if more than one is present - the staining characteristics (gram positive or gram negative) - the shape of the organisms, rods (bacilli) or round (cocci) - the size of the organisms: small, large, thin, plump - the configuration: single organisms, pairs, chains, clumps, clusters, branching - the relation to inflammatory cells because some organisms are characteristically inside inflammatory cells (intracellular) or adherent to them