Laboratory Diagnosis and Bacterial Identification Flashcards
What are Characteristics of Light Microscopy?
Most Common Type
Resolution = 0.2um
Microbes larger then virus
Wet Mounts to Study Live Microbes
- ) Fluids (Urine ,CSF or Feces)
- ) Larger Cells ( Parasites , eggs and protazoa)
- ) Fungi from Skin (KOH)
What are the Stained Preparations of Light Microscopy?
Gram Stain
Acid Fast Stain ( Kinyoun , Ziehl Neelsen)
Capsule Stain ( India Ink)
Spore Stains
Giemsa or Giemsa Wright
Silver Stains
What is the Principle of Darkfield Microscopy?
The light microscope is adapted by modifying the condenser to prevent transmitted light from directly illumintating the unstained species
Light Passes around (reflected) rather then through
Spirochetes appear bright (thin)
What is the Principle of Phase Contrast Microscopy?
Unstained sample
Uses light refraction to give 3D image
Better visualization of Internal Structures
What is the Principle of Fluorescence Microscopy?
Fluorescent Compounds
Absorb Energy at One Wavelength and Emit
Staining uses Calcofluor White (Fungi) or Auramine O Rhodamine (Myobacteria)
Targeted Fluorophores are conjugated to Antibody
Direct (AB to Antigen)
Indirect ( Secondary AB to AB to Antigen)
What is the Principle of Electron Microscopy?
Can visualize smaller particles
Virus and Organelles
Scanning EM gives a surface image
Transmission EM uses stain to provide internal image
What is a Blood Agar Media (BAP)?
General Purpose and Differential Medium
Sheep Blood
Growth Factors for Fastidous Organisms ( Complex Requirement)
Pattern of Hemolysis can aid in Identification
What type of Media is Shown in the Image
Blood Agar
What is the characteristics of a Choclate Agar (CAP) and Thayer Martin Agar?
Contains blood that has been heated to lyse red blood cells and release intracellular nutrients
Organisms with difficult nutritional requirements that cannot lyse themselves
Haemophilus and Neisseria
Thayer Martin has antibiotic added for isolation of Neisseria Gonorrhoeae
What type of Media is Shown in this Image?
Choclate Agar or Thayer Martin Agar
What are the Characteristics of a Mannitol Salt Agar(MSA)?
High Salt Concentration inhibits all but osmotolerant organisms
Fermentation of Mannitol results in acid production , causing a drop in pH and turning pH indicator from red to yellow
Staphylcocci
Differentiating Staph Aureus (Ferments Mannitol) form Coagulase negative Staph
What type of Media is Shown in the Image?
Mannitol Salt Agar (MSA)
What are the characteristics of the MacConkey and Sorbitol MacConkey Agar?
Contains Bile Salts and Crystal Violet to inhibit growth of Gram Positive Organisms and some Gram Negative
Selective for Gram Negative Bacteria
Lactose and Neutral Red pH Indicator
If Organism ferments lactose , colonies appears pink (Low pH) (E Coli)
If Organism is non lactose , colonies appear beige (Salmonella)
Can use Sorbitol as Well
What type of media is shown in the image?
MacConkey Agar (MAC)
What are the Characteristics of Eosin Methylene Blue Agar?
Use Eosin and Methylene Blue to inhibit Gram Positive
Lactose Fermenters appear Blue/Black
Non Fermenters are Clear
E Coli has a shiny green
Similar to MAC
What type of Media is Shown in the Image?
Eosine Methylene Blue Agar
What is the characteristics of a Bile Esculin Agar (BEA)?
Bile Salts Inhibit the growth of Gram Positive Organisms and most Streptococci except Group D Streptococcus and Enterococcus
Esculin cany by hydrolyzed by Group D to Esculitin which interacts with iron to form a black precipitate or ferrous sulfate
What type of media/tube is shown in the Image?
Bile Esculin Agar (BEA)
What is a Hektoen Enteric Agar (HE)?
Bile Salts and Indicator to Inhibit Gram Positive Organisms
Use Lactose , Sucrose and Salicin
Fermenters apper Yellow Orange
Non Fermenters Appear Green or Transparent
Hydrogen Sulfide (H2S) producers will form Black Precipitate
Salmonella
What type of Media is shown in the image?
Hektoen Enteric Agar (HE)
What are the Characteristics of a Cetrimide Agar?
Isolation of Pseudomonas Aeruginosa
Detergent that Inhibits most bacteria
Contain magnesium and potassium which promote the produciton of the pigments pyocyanin (Blue Green)
Fluorescein ( Yellow Green)
What type of media is shown in this Image?
Cetrimide
What are the Characteristics of Tellurite Agar? (Cystine Tellurite Agar)
For isolation of Corynebacterium Diphtheriae
Potassium Tellurite inhibits Gram Negative Organisms and most Upper Respiratory Flora
Colonies appear gray or black (tellurite reductase activity) with a brown halo (cysteinase activity)
What type of media is shown in this image?
Tellurite Agar
What are the Characteristics of a Triple Sugar (TSI Agar)?
Contain Lactose , Sucrose , Glucose , Protein Digest , Sodium Thiosulfate , Ferrous Iron and a pH indicator
Pink to Yellow Color Change Indicates:
A.) No Yellow , No Sugar Utilization
B.) Entire Tube Pink , Multiple Sugars
c.) Glucose Fermentation , Pink on Top with Black (H2S)
D.) H2S Produciton of a Black Precipitate
What type of tube test is shown in the Image?
Triple Sugar Iron Agar
What are the Characteristics of a Chromogenic Agars?
Change in colony color indicates different colonies
Specific and Grow Quick
Listeria Monocytogenes
Staphylococcus Aureus
Methicillin Resistant vs Methicillin Sensitive
What type of Media is shown in this Image?
Chromogenic Agar
What are the three types of Hemolysis Reactions on a Blood Agar plate?
Alpha Hemolysis
Partial Lysis of the RBCs
Organsims produce Hydrogen Peroxide
Oxidation of Hemoglobin to Green Methemglobinperoxide
Beta Hemolysis
Complete clearing of the RBC
Organsims produce Hemolysin which Lyses
Gamma Hemolysis
Bacteria have no Effect on the RBCs
What is a Catalase Test?
Detects presence of Catalase Enzyme
Converts Hydrogen Peroxide to Water and Oxygen
Differentiate Gram Positive Bacteria
Anaerobes Less Likely
Rapid Under a Minute
What is a Coagulase Examination?
Detects Bacteria which convert fibrinogen to fibrin
Staphylococcus Aureus
Yersinia Pestis
Free Coagulase (Overnight)
Secreted bacterial coagulase reacts with prothrombin in the liquid test tube medium to form staphylothrombin which catalyzes the conversion of fibrinogen within the medium to insoluble fibrin
Medium becomes a Gel
Bound Coagulase
Bound to bacterial cell wall
Done on Slide (Few Minutes)
What is a Fermentation Test?
pH indicator in media reflects the production of acid from metabolism of a sugar in a medium
Differentiate gas productions vs none
Home/Hetro Fermentation
Happens Overnight
What is an Oxidase Test?
Detect the Presence of Cytochrome C Oxidase
Indicator turn Purple
Some bacteria with ETC have a different molecule and will test negative
Helps Differentiate Gram Negative Bacteria
Rapid
What is an Urease Test?
Detects the presence of the urease enzyme
Breaks down Amides which produces ammonia
Result in a Color change to pink
Can be agar slant or broth
Helicobacter , Proteus , Ureaplasma , Nocardia and Cryptococcus
What is a Thioglycollate Test?
Thioglycolates Remove Oxygen
Bacteria places in semi solid test tube allowing oxygen to diffuse through the top
Creates and Oxygen Gradient:
- ) Obligate Aerobes at the Top
- ) Aerotolerant Anaerobes throughout the tube
- ) Strict Anaerobes in the Bottome
Overnight
What is a Direct Assay? (Latex Agglutination Test)
Determine the presence of Antigens
Inert latex beads conjugated to specific antibodies are mixed with a specimen
Results in a Visible Clumping Reaction
IgM antibodies work the same way
Results within Minutes
Non Specific Binding and Low Sensitivity
Qualitative
What is a Sandwhich Enzyme-Linked Immunosorbent Assay (ELISA and EIA)?
Use of Enzymatic Detection mechanism or Fluorophore can amplify the signal , providing high levels of Sensitivity
The amount of signal is proportional to the amount of detection present , which is proportional to the analyte
Compare to a Standard Curve
What is a Chromotographic Immunoassay (CIA)?
Dipstick or Strip Format
Capillary action brings a dye conjugated antibody antigen complex to a second , immobilizded capture antibody
Leaves a Concentration Dot
What are Nucleic Acid Based Tests?
Detection of Specific Nucleic Acid Signatures via Hybridization to complementary nucleic acid sequences
Use PCR or Branched DNA Assay to Amplify
Reverse Transcriptase PCR
Starts from RNA , RT enzyme converts the RNA into complementary DNA (cDNA) , then normal PCR used
Can be Real Time as Well (Fluorescence)
Used for RNA Viruses
Real Time PCR
Incorporates visualization of the product via fluoroscence as amplification occurs
What is Branched DNA Assays?
Amplifies the Signal not the Nucleic Acid itself
Single Stranded DNA probe is generated that incorporates a portion that is complementary to the targeted sequence and a portion that will bind an engineered branched DNA Structure
Conjugated to Enzymes that will create a signal when mixed with a substrate
Single Nucleic Acid Signal will produce a highly amplified signal , and the amount is proportional to the number of copies of the specific nucleic acid target
What are some Advantages and Disadvantages to Serologic Tests?
IgM Indicates an early active Infection
IgG takes several weeks (Active or Previous Infection)
Advantage
Usually Very Sensitive
Acute Infection
Disadvantage
IgM antibodies can only be detected early in infection
IgG Antibodies appear 2-4 weeks after
What is Indirect ELISA?
Test kit contain antigen from the organism of interest
Patient serum is applied and wells/slides are washed
If antigen specific antibodies are present they will bind and will not wash off
ELISA is enzyme linked while IFA is Fluorophore
Second Antibody will bind them and will remain through a wash step
Label permits detection
Subject to Lab Techs Experiance
What is a Western Blot?
