Culture and ID Methods

What are the phenotypic characteristics of bacteria that can be observed?

• colony morphology

• microscopic morphology

• staining characteristics

• environmental requirements for growth (oxygen or no oxygen)

• nutritional needs

• biochemical characteristics (catalase, oxidase, etc.)

• antimicrobial susceptibility

When observing colony morphology, what is noted about the colonies?

• hemolysis patterns

• size

• form/margin

  • what the edges of the colonies look like

• elevation

• surface consistency

  • mucoid, creamy, viscous, brittle, dry, etc.

• density

• color

What type of hemolysis pattern is this?

beta

What type of hemolysis pattern is this?

alpha

What are the 5 margins of bacterial colonies?

smooth (entire), rough, irregular, curled, and filamentous

• page 85 in textbook

What are the 5 types of elevation of bacterial colonies?

flat, convex (dome shaped), raised, umbilicate, and umbonate

List the 6 preliminary biochemical tests used for bacteria identification

• carbohydrate utilization

• catalase

• coagulase

• cytochrome oxidase

• spot indole

• PYR hydrolysis

Carbohydrate utilization test

• enzymes within specific bacteria can break down carbohydrates in the absence of oxygen = fermentation

• carbs broken down in the presence of oxygen = oxidation

• growth media contain carbohydrates and an acid base indicator for identifying if the organism uses a certain carbohydrate

  • if a carbohydrate is utilized, a color change will occur

  • glucose, lactose, maltose, sucrose, and fructose

catalase testing

• some organisms produce the enzyme catalase, which helps them survive phagocytosis by breaking down hydrogen peroxide into water and oxygen bubbles

• catalase positive = bubble formation

• catalase negative = no bubbles

• can help distinguish between Staphylococcus and Streptococcus

  • Staph is positive, Strep is negative

coagulase testing

• the enzyme coagulase converts fibrinogen to fibrin

• Staphylococcus aureus produces coagulase

• slide test

  • colony is mixed with rabbit plasma, positive reactions indicated by fibrin clots

• tube test

  • rabbit plasma is mixed with the organism and incubated

cytochrome oxidase test

• detects if an organism produces cytochrome oxidase enzyme for metabolism

• an oxidase reagent and the specimen are placed on filter paper

• positive reactions are purple

• oxidase positive genuses:

  • Neisseria, Pseudomonas, Campylobacter, Aeromonas, and Pasturella

  • not common in the Enterobacteriaceae family

spot indole test

• detects if an organism has the enzyme tryptophanase

  • breaks down tryptophan when bacteria are grown on SBA or chocolate agar

• a colony is smeared onto filter paper and Kovach’s reagent is added

  • positive reactions are blue-violet

• differentiates E. coli (indole pos) from other Enterobacteriaceae, and Proteus mirabilis (indole neg) from Proteus vulgaris (pos)

PYR hydrolysis test

• detects if bacteria have the ability to break down the substrate PYR

• important for differentiating:

  • Group D enterococcus (pos) from non-enterococcus (neg)

  • and group A Streptococcus (pos) from group B Streptococcus (neg)

• positive reactions are pink

What are multitest systems?

• single-step systems that incorporate inoculation, incubation, interpretation of results, and reporting of results for specimens

• biochemical and enzymatic reactions

• can be manual or semi-automatic

• uses numeric codes and databases based on the biochemical profile of microorganisms

What are the advantages of multitest systems in the micro lab?

• shorter incubation and reaction times

• reproducible, yields consistent results

bioMerieux API multitest system

• utilizes microtubes with dehydrated substrates that are reconstituted by adding a bacterial suspension

• allows for the identification of:

  • Enterobacterales, Streptococcus, Staphylococcus, Neisseria, Haemophilus, Corynebacterium, anaerobes, non-fermenters, and yeast

• a large database for the organism profiles is accessible through the internet-based API web service

How does the bioMerieux API multitest system work?

• after incubation for 18-24 hours, reactions are read, and a profile number is determined

• determining the organism’s profile number:

  • the 20 microtubes are broken down into groups of 3

  • the microtubes with positive reactions are assigned points and the points are added up for each group

  • the 7-digit code of all the added up points can be entered into the database for microbial ID

What are the automated ID systems for detecting metabolic activity in organisms?

• Vitek 2 by BioMerieux

• Microscan Walkaway by Beckman Coulter

• Sensititre by ThermoScientific

• Phoenix by BD

In detecting metabolic activity, organisms are identified by their utilization of substrates. What are the 3 ways to detect end products in microbial metabolism?

• colorimetry

  • a spectrophotometer measures color change using pH indicators or substrates

• nephelometry

• fluorometry

nephelometry

• measures light scatter

• useful for antibiotic susceptibility testing

  • wells are coated with antibiotic

  • as an organism grows, the substrate becomes more turbid

  • the more turbid the solution is, the more resistant the bacteria is to the antibiotic

fluorometry

• uses biochemical substrates bound to a fluorescent component, such as a fluorophore

• if the organism has an enzyme to metabolize the substrate, the fluorophore is released and fluoresces

What does MALDI-TOF MS stand for?

Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

How does MALDI-TOF MS work?

1. a specimen is applied to a target plate

2. matrix (a chemical) is added to the target

3. a laser hits the target and ionizes the sample

4. ionized molecules fly up through a vacuum tube and hit a detector (time of flight is the time in nanoseconds for ion travel)

5. a spectrum is generated based on signal intensity

   1. this spectrum is unique to each species and is compared to the spectra of known organisms in a database for identification

What are the pros and cons of MALDI-TOF MS?

• pros

  • quick results

  • cost effective

  • yields accurate and reproducible results

• cons

  • identification is limited to the database

  • only pure cultures can be used

  • cannot be used with clinical specimens (due to contaminants or multiple organisms)

How do automated blood culture systems work?

by measuring the log phase of growth of microorganisms in blood cultures