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Bio safety Level 1

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Agents have no known potential for infecting healthy people

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Bio safety Level 2

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i. Most clinical labs fall into this category
ii. Agents handled include all the common agents of infectious disease as well as HIV
iii. Requires limited access and training of laboratory personnel
iv. Hep B vaccine must be offered to all employees

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bio safety Level 3

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i. Agents handled are unlikely to be handled in the routine clinical lab
ii. Mycobacteria, mold stages of systemic fungi and other highly infectious agents
iii. Special room and ventilation required as well as special protective clothing

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bio safety Level 3

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i. Level 3 plus entrance through a separate room. Street clothing changed and replaced with laboratory clothing. All personnel and materials are decontaminated before leaving the area
ii. Primarily research and public health facilities such as the CDC

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Biosafety Cabinets Class I

answer

allows room air to flow into the cabinet and around the area and material within

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Biosafety Cabinets Class 2

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Sterilize both the air that flows over the infectious material and the air that is exhausted.
Generally used in most hospital laboratories.

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Biosafety Cabinets Class 3

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Level II plus it is a closed system where rubber gloves are used in handling samples. There is no
direct contact from outside the system. Generally used in Biosafety level 4 labs

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Nomenclature 1

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The naming of microorganisms according to established rules and guidelines provides the accepted labels by which organisms are universally recognized. The genus and species of an organism are of most concern to microbiologists.

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Nomenclature 2

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i) By convention the genus is always Capitalized and the species is not
ii) Both are either underlined or italicized

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Identification Genotype

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relates to an organisms genetic makeup such as DNA or RNA

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Identification Phenotype

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Macroscopic morphology – Growth patterns on artificial media
ii) Microscopic morphology – Size, shape, appendages etc.
iii) Staining characteristics – Ability of organism to stain a particular color
iv) Environmental requirements – Temperature requirements
v) Nutritional requirements – Carbon and nitrogen utilization patterns, carbohydrate fermentation
vi) Antigenic properties – profiles by serologic methods
vii) Subcellular properties – Cell wall and membrane components

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Coccus

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i. Plural – Cocci meaning berries
ii. Diplococci – pairs
iii. Streptococci – chains
iv. Tetrads – groups of 4

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Bacillus

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i. Plural – bacilli meaning little staffs
ii. Diplobacilli – pairs
iii. Streptobacilli – chains
iv. Coccobacilli – Bacilli that are oval and may appear more like Cocci

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Spiral

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i. Vibrios – Bacteria that look like curved rods
ii. Spirilla – helical or corkscrew shape and are rigid
iii. Spirochetes – Helical or corkscrew shape and are flexible

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Gram positive Cocci

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o Staphylococcus
o Streptococcus
o Enterococcus

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Gram Negative Cocci

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o Moraxella catarrhalis – diplococci
o Neisseria gonorrhoeae – diplococci
o Neisseria meningiditis - diplococci

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Gram negative Bacilli

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o Bacteroides
o Camplyobacter
o Escherichia coli
o Klebsiella
o Salmonella
o Shigella

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Gram positive Bacilli

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o Actinomyces- branched
o Bacillus- long wide, B. anthracis- “safety pins”
o Clostridium
o Corynebacterium ( diphtheroids)- palisades or Chinese letters o Listeria

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Gram Variable

answer

o Gardnerella vaginalis - coccobacilli

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Cellular appendages-Capsule

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Exterior to the outer membrane. Referred to as the slime layer, or glycocalyx, is composed of
high molecular weight polysaccharides. Protects bacteria from phagocytic attack by cells of the immune
system and increases adherence of colonization surfaces

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Cellular appendages-Fimbriae or pili

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long hair like structures that extend from the cell membrane. Help attach to host cell
surfaces

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Cellular appendages-Flagella

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complex structures embedded in cell envelope. Responsible for cell motility if present

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Endospores

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Under adverse conditions some bacteria are able to form spores. Involves from an active cell to a dormant cell with a decrease in cytoplasm and an increase in the thickness of the cell envelope. Frequently found in microbes living in soil such as B. anthracis

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Normal Flora

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Microorganisms that are commonly found on or in body sites of healthy persons
a. Different body sites have different or the same flora depending on the local conditions

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Resident microbial flora

answer

colonize an area for months or years

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Transient microbial flora

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temporarily present at a site, does not normally occur or stay at a particular site. Usually eliminated by immune response or competition w other normal flora

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Carrier

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Host person who has established pathogenic flora with no disease present, they are however capable of transmitting disease

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Carrier State

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The condition of being a carrier for a pathogenic organism
a. May be Acute, short lived or transient
b. May be Chronic, lasting months, years or permanently

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Factors that Affect Normal Flora-Environmental Factors

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a. Factors such as temperature, moisture.
b. pH of the site has a great effect on colonization, as in the female genital tract where the normal pH in women of child bearing age is a very acidic 4.0-5.0. Most bacteria cannot survive in an environment with such a low pH.

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Factors that Affect Normal Flora-Nutritional Factors

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a. Presence of chemicals unique to certain body sites and uniquely used by certain species of organisms. presence of chemicals such as lipids (fats), bile, lysozymes, and fatty acids all affect the composition of organisms colonizing each area individually. Ex. Propionibacterium acnes colonize the ducts of hair follicles because it is able to break down the skin lipids into fatty acids

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Sterile Body Sites- Lower Respiratory Tract(were specimen and testing for)

answer

Trachea, bronchi and lungs a. Specimen- Sputum, bronchial lavage
b. Pathogens
I. Streptococcus pneumonia
ii. H. influenzae

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Sterile Body Sites-Upper Genitourinary Tract-(were specimen and testing for)

answer

a. Specimen – Cath Urine, Surgical specimens
b. Pathogens- generally coliform bacteria cause infections of the urinary tract
i. E. coli
ii. Proteus mirabilis
iii. Staph saprophyticus

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Sterile Body Sites-CNS-(were specimen and testing for)

answer

Brain, spinal cord and Cerebral Spinal fluid a. Specimen – CSF cerebral spinal fluid
b. Pathogens
i. E. coli
ii. Neisseria meningitis

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Sterile Body Sites-Peritoneal Cavity(were specimen and testing for)

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(abdominal cavity) a. Specimen – Ascites fluid
b. Pathogens – Coliform bacteria from GI tract

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Sterile Body Sites-Synovial capsules(were specimen and testing for)

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( fluid filled joints) a.Specimen – synovial fluid
b. Pathogens
i. N. gonorrhoeae
ii. Staphylococcus aureus

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Sterile Body Sites-Blood(were specimen and testing for)

answer

a. Specimen – blood cultures are collected directly from the blood stream into enriched broth for incubation b. Pathogens – Many
i. E. Coli, Staph aureus
ii. Almost any infection site can become systemic when the bacteria from an infection site enter the blood stream and multiply
iii. Condition known as Sepsis or Septicemia indicated by abrupt onset of fever and chills
iv. Can be Aerobic or Anaerobic bacteria

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Laboratory diagnosis of infectious diseases involves

answer

? Direct examination of patient specimens for presence of etiologic agents ( bacteria, fungi) and for
sample integrity.
o Direct exam of a wet prep mount of vaginal secretions for report of white blood cells,
trichomonas, yeast cells and clue cells
o KOH prep – used to dissolve skins cells and detect the presence of fungal elements
? Isolation, growth and cultivation of the microbes
? Analysis of the cultivated organisms to establish identification

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Simple Stains

answer

Methylene blue – imparts the same color to all parts

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Differential Stains

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Gram Stain
o Acid-fast Stain – Used to Stain Mycobacteria o Capsular Stain –
o Endospore Stain
o Flagella Stain

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Gram Stain

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? Primary Stain – crystal violet
? Mordant – iodine
? Decolorizer- alcohol or acetone ? Secondary Stain – Safrinin
Gram positive organisms retain the purple crystal violet coloration
Gram negative organisms release the crystal violet/iodine complex and stain pink with the Safrinin White blood cells appear pink to reddish orange in a gram stained specimen as opposed to the purple color seen in a Wright Stained blood smear

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Negative Stain

answer

India Ink – dark background for use in visualizing the capsule surrounding certain organisms such as the
yeast Cryptococcus which has a large capsule

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Specimen Collection and Handling-Wounds

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a) Swabs are easily contaminated and dry out quickly
b) If used, usually are placed in transport gel to retain specimen integrity
c) Deep wound or abscess specimens are best collected by needle aspiration, NOT SWABS
d) Note the type of sample and the anatomical site of collection

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Specimen Collection and Handling-Urine

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clean-catch or nurse collected catheter specimen

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Specimen Collection and Handling-Sputum

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a) Sputum samples may be refrigerated for transport in a sterile container
b) Sputum vs. Saliva
i) Sputum samples are gram stained upon receipt in the lab to determine specimen integrity. Specimens with >24 epithelial cells and multiple organisms are generally rejected due to contamination with oral flora.

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Specimen Collection and Handling-Spinal Fluid

answer

suspected meningitis
a) Should be handled stat and processed immediately.
b) Gram stain always performed, normal specimen is sterile, no organisms seen
c) Cultures are performed with specimen inoculated to chocolate agar and thio broth to enhance chances of
recovering fastidious organisms
d) May be held at 370c up to 6 hours prior to culture if needed

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Specimen Collection and Handling-Fecal Specimens

answer

a) Collected to diagnose GI pathogens such as Salmonella and Shigella, common food poisoning assoc. organisms
b) Preserve at 7.0pH if testing is to be delayed
c) Special preservative vials are used for O&P samples to preserve those organisms

question

Specimen Collection and Handling-Blood Cultures

answer

collected directly into broth bottles
a) Used to diagnose Bacteremia or Septicemia
b) Sterile technique is imperative in collection of samples
c) Normally collected at least two times form two different sites
d) Avoid refrigeration

question

Specimen Collection and Handling-Throat

answer

a) Usually 2 swabs are collected, one may be used for rapid testing of Group A Beta Strep (S. pyogenes)
b) The Second swab is used for culture
c) Transport within one hour

question

Specimen Collection and Handling-Anaerobic organisms

answer

Some are very sensitive to environmental changes and will not be recovered on culture if not handled properly.
a) Obligate Anaerobes – cannot live in the presence of oxygen.
i) Specimens for anaerobes must be places immediately into anaerobic transport gel to preserve those
organisms
b) Facultative Anaerobes – many organisms isolated from the GI tract are facultative anaerobes. They can grow
with or without the presence of oxygen.

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