2nd practical

genus streptococcus

• coccus - in chains

• gram positive

• no enzyme catalase

• fastidious microaerophilic organisms

  • Need extra nutrients for growth

  • More CO2 and less O2 than atmospheric level

streptococcus pathogenic

• normal throat flora and beneficial active cultures

• Strep Throat, Pneumonia, meningitis, endocarditis, pharyngitis

streptococci species identification methods

• hemolytic activity

• lancefield classification system - cell surface antigens

hemolytic patterns

• beta

• alpha

• gamma

hemolysis

Ability of bacterial enzymes to cause lysis of the red blood cells when grown on blood agar

beta - hemolysis S. pyogenes

• complete destruction of red blood cells (and digestion)

• Results in a clear area around the bacterial colony

alpha - hemolysis S. pneumoniae

• partial hemolysis (no digestion)

• Hemoglobin is modified to form hemoverdin

• Produce greenish/brownish zone around the colonies

gamma - hmolysis non pathogenic Enterococcus faecalis

• absence of hemolysis - no reaction surrounding the colony

• No bacterial enzymes to lyse red blood cells

lancefield classification

• based on unique proteins (antigens) on cell surface of streptococcus species

• pioneered by rebecca lancefield

• separates streptococcus into 13 groups (A,B,C,D….)

  • A-D cause most human disease

Group A Streptococcus

• Sensitive to Bacitracin (ZOI around antibiotic disk using KB method)

• Beta hemolytic/complete clearing

• Only 1 species - Streptococcus pyogenes

  • Causes Strep throat > Untreated > secondary infections

  • Group A can be ‘flesh-eating’ or ‘pus generating’

Group B Streptococcus

• agglutination Test

• Beta hemolytic/complete clearing

• Only 1 species – Streptococcus agalactiae

• normal flora of the vaginal mucosa but can be severely harmful to babies when they are born

  • Causes neonatal meningitis and septicemia

Group D Streptococcus

• may be alpha, beta, or gamma hemolytic

• Includes many different species

  • Enterococci >> E. faecalis endocarditis, biliary infections, UTIs = VRE

Streptocard Acid Latex Test

• Rapid test for Lancefield grouping

  • Does NOT work for Streptococcus pneumoniae

• Antibody-Antigen reaction - agglutination

  • A reagent with latex beads coated with a specific Lancefield

    antibody.

  • Presumably only one antibody would recognize the antigen of

    Streptococcus.

  • Agglutination occurs when the antibody coated latex beads

    specifically bind to the specific antigen and crosslinks with

    multiple latex beads.

    • Result is visual clumping of the latex beads

Lancefield Strep Typing

• Latex beads coated with a specific Lancefield antibody

• Agglutination occurs when the antibody coated latex beads bind to the specific antigen resulting in crosslinks of multiple latex beads (visible clumping occurs)

Agglutination

Rapid Antibody-Antigen reaction test for Lancefield grouping

Staphylococcus vs Streptococcus

Staphylococcus - occurs in clusters, Catalase positive, Coagulase positive, Protein A - surface protein, Tests: Staphyloslide (quick), Coagulase test (incubate)

Streptococcus - occurs in chains, Catalase negative, lancefield classification system, tests: lancefield strep typing (quick), hemolysis typing (incubate)

biochemical testing and identifying bacteria

reals information necessary to help identify bacteria within a sample

metabolism

used as an additional factor to identify bacteria because many of them share colony and cell morphology. bacteria produce enzymes that play a role in metabolic processes. the enzymes allow for identification through biochemical testing

biochemical testing agar

Simmons citrate agar and urea agar

Simmons citrate

• differential

• enzyme citrate lyase are able to use citrate as a carbon source

• when citrate is used it becomes more alkaline

  • turns green to blue

Bromothymol Blue

the pH indictor in the medium for a Simmons citrate agar, changes medium from a green to blue color.

Uninoculated Simmons Citrate slant

pH 6.9 – 7.6, green agar slant, negative reaction

Inoculated Simmons Citrate slant

pH > 7.6, blue agar slant, positive reaction

urea agar

• differential - only organisms with enzyme urease can break down urea

• when ammonia (NH4) is freed from the agar it causes a pH change and the environment becomes more alkaline

urea

a waste product of protein digestion in most vertebrates and is excretes in the urine

pH indicator in urea agar

phenol red, changes color yellow to hot pink (fuchsia)

Uninoculated Urea agar slant

pH 6.8 – 8.0, yellow agar slant, negative reaction

Inoculated Urea agar slant

pH > 8.0, fuchsia agar slant, positive reaction

2 differential media

contains various nutrients that allow one to distinguish one bacterium from another by how they metabolize or change media with a waste product

mannitol salt agar

• selective = contains a high salt concentration (only organism that are halophiles or halotolerant will grow)

• differential = contains the sugar mannitol (looking for mannitol fermentation)

• also contains pH indicator called phenol red

fermentation of mannitol

the medium will change from red to yellow due to the pH

pH color change for mannitol

pH >8.4 = pink

pH 6.9-8.4 = red

pH < 6.9 =

Microbiological Culture

method of multiplying microbial organisms by letting them reproduce in a predetermined culture media under controlled laboratory conditions

mixed culture

yellow, more than one type of organism

pure culture

pink, single type of organism, main idea is to dilute the original sample until the organism of interest is isolated and pure

ways to obtain pure culture

spread, pour, streak plate

details of methods of getting pure culture

they dilute or thin out a heavy population of bacteria across an agar surface. Once a pure culture is obtained it can be used to identify if the bacteria is sensitive or resistant to an antibiotic

spread plate

original culture is serially diluted then the final dilution is spread on the surface of the plate. surface colonies grow

pour plate

serial dilution than final dilution added to molten agar which is poured over an agar plate. surface and subsurface colonies grow

streak plate

original culture directly diluted across (agar) new plate with inoculating loop. 3 sections in a T pattern, in each section start with a line from the previous section

types of media

broth, agar

broth

liquid media; nutrients (used in motility experiment)

agar

jellylike substance derived from seaweed: thickening agent

why do we use agar

because microorganisms cannot digest agar. it’s a solid surface for microorganisms to grow and we can pick out individual colonies

all purpose/supportive media

contains nutrients that will support the growth of a large variety microorganisms

selective media

promote growth of some bacteria and/or limits growth of other bacteria

differential media

distinguish between different bacteria based on changes in colonies or changes in media

types of agar

tryptic soy agar, Eosin methylene blue agar

tryptic soy agar (TSA)

all-purpose/supportive medium used to grow most microorganisms

Eosin methylene blue agar (EMB)

selective media for gram negative organisms. inhibits the growth of gram-positive organisms due to the dye’s eosin Y and methylene Blue

lactose fermentation makes it a differential media. it causes precipitation of the dyes on the surface of the colonies resulting in different colors.

Lots of acid = green metallic sheen

small amount of acid = pink or blue center (fish eye)

no fermentation = colorless

aseptic transfer

conducting your work in a way that will not contaminate the culture itself, and also without contaminating your workspace to yourself with the specimen

keep the lid on the plate at all times, can come off to pick a colony and then immediately

how to achieve aseptic technique

• disinfect work area

• all tools that handle bacteria need to be sterile

  • loops/needles: flamed in the incinerator or Bunsen burner to sterilize

  • tubes, plates, etc: autoclaved

• keep all cultures covered unless you are using it that second

• if unaware if tools are sterile start over