Credit Test 1

• morphology

• gram staining ability

• acid resistance properties

• presence of capsule

• presence of endospore

• motility

• quanitity of microscopic elements

• placement of bacteria (extra/intracellular)

• presence of cellular elements (epithelilal cells,..) —> appropriate collection is important

• inflammatory activity —> presence, type + amaount of leukocytes

if presence of spirochetes is suspected

• size

• shape

• arrangement

• movement

• stabilizes smear

• inactivates most bacteria (kills vegetative forms)

• improves staining ability

only 1

• methylene blue

• carbofuchsin

• presence

• quantity

• shape

• arrangement

• weak alkalic solution of dye —> crytalline violet

• alkaline Lugol´s solution —> stabilized dye bond

is based on speed of decolarization of bacteria by non-polar solvents (acetone, ethanol)

• Gram negative: good penetration through cell wall —> rapidly decolorizing

• Gram positive: decolorize after a long period of exposure

—> contrasting “secondary” complementer counter staining (carbolfuchsin, safranin) —> visualize the decolorized gram-negative microorganisms

• primary staining —> crystalline violet

• add Lugol´s solution for 2min

• decolorize by acetone

• rinse with water

• secondary stain —> csrbofluchsin

• rinse with water

• dry

• observe 1000x magnification + immersion oil

positive:

• Staphylococcus aureus

• Candida albicans

• Streptococcus pneumoniae

negative

• Eschericia Coli

• Klebsiella pneumoniae

• Haemophilus influenzy type b

  
  

• without peptidogycan cell wall (Chlamydia, Mycoplasma)

• with thin cell wall (spirochetes)

• acid-fast bacteria (mycobacteria)

• endospores of bacteria (Bacillus, Clostridum)

• Intracellular bacteria (Ricketsia, Ehrlichia)

Ziehl-Neelsen staining

• for acid-fast bacteria —> they posses lipid-rich cell wall (waxy substances —> mycolic acids)

• are resistant agains —> alcali, alcohol, acid (AAA resistance)

• Mycobacterium spp.

• Nocardia spp.

• Actinomyces spp.

• stain with concentrated carbolfuchsin + heat above flame until vapor arises (repeat this step at least 3x)

• decolorize by acid alcohol

• rinse with water

• stain by methylene blue

• rinse with water

• dry

• observe

• we can find slim rods with various length that can form aggregates

• we observe 200 dields of view in the preparation

• evaluation is done semiquantitatively —> 0 cells (-), 1-20 cells (+), 21-100 cells (++) more than 100 (+++)

• visualization of a capsule

• capsule = polysaccharidic substance that protects bacteria from immune response (phagocytosis) —> encapsulated cells are more virulent

• E. Coli K1

• Haemophilius influenza type b

• Streptococcus pneumoniae

• Klebsiella pneumoniae

• prepare suspension —> create smear —> fix smear by heat

• stain by diluted carbolfuchsin

• rinse

• dry

• observe

visualization of endospores

endospores:

• can survice many years in environment (but destroyed by termperature of 121°C for 20min under pressure)

• have extraoridinary resistant to many physical + chemical agents —> resistant to drying, ultraviolet rays, higher temperature

• can only be stained by concentrated dyes by using heat

• after colorization it is difficult to decolorize them

• Bacillus spp. (Bacillus cereus, Bacillus anthracis)

• Clostridium spp. (Clostridium botulinum, Clostridium tetani)

• fixed preparation by heat

• concentrated malachite green —> heat until vapor arises (repeat step at least 3x)

• rinse

• stain with diluted carbolfuchsin

• rinse

• dry

• observe

• used to visualize epithelial cells, leukocytes, cellular structures

• staining of parasites (parasitic protozoa —> Trichomonas vaginalis, Plasmodium spp.)

• staining of intracellular bacteria (Chlamydia trachomatis, Mycoplasma spp.)

• preperation fixed chemically by methyl alcohol

• stain by “Giemsa dye” (30min)

• rinse

• observe

procedure that provides growth + multiplication of microorganisms in laboratory conditions

• natural —> contain components from plant + animal source (blood agar)

• synthetic —> contain chemically defined compounds

• semi-synthetic —> contain elements of both

• liquid

• semi-solid

• solid

• basic (universal) —> blood agar

• diagnostic media —> conatin suitable indicator ehich reveals certain metabolic or physiologic properties of tested microorganism (McConkey agar, blood agar)

• selctive media —> contain substance that can supress the groeth of certain microorganism but targeted microorganism gains an advantage of elective groeth (Clauberg agar, Dezoxycholate agar, MacConkey agar)

• special —> for specimen contaminated by flora (selenite broth)

• special-enriched —> for fastidious bacteria (chocolate agar)

• for antibiotic susceptibilty testing —> Müller-Hinton agar)

• for cultivation of fungi —> Sabouraud agar

• for cultivation of some parasites —> diamond medium

• blood agar —> fastidious baceria + evaluating hemolysis

• chocolate (levintha´s) agar —> hemophilis + pathogenic neisseria

• Clauber´gs agar —> selective for corynebacteria

• Selentine agar —> selective for salmonella

• endo agar —> enterobacteriacae, lactose fermentation testing

• desoxycholate agar —> enterobacteriacae, lactose fermentation + hydrogen sulfide testing

• MacConkey agar —> enterobacteriacae, lactose fermentation testing

• Müller-Hinton agar —> antimicobials susceptibility testing

• Sabourrauds agar —> yeast + molds cultivation

• sediment (Streptococcus salivarius)

• membrane (Pseudomonas aeroginosa)

• turbidity (E. Coli)

b-hemolysis —> blood agar gets transparent

a-hemolysis —> greenish color because hemoglobin changes to verdoglobin

gamma-hemolysis —> No hemolysis

• Staphylococcus aureus

• Streptococcus pyogenes (strong)

• Streptococcus agalactiae (weak)

  
  

• Streptococcus pneumoniae

• Streptococcus salivarius

Staphylococcus epidermidis

• VL agar

• Schaedlers´agar

• thioglycolate medium

Haemophilius influenza

• Levinthal agar

• No —> blood + McConckey agar

Staphylococcus aureus

• blood agar + levinthal agar

• NO —> McConckey agar

Escherichia coli

• blood agar, levinthal agar, McConckey agar

• Biological —> growth, morphology of colonies, shape of cells, staining properties

• Biochemical —> detection of enzymatic properties of bacteria by biochemical tests

• Serological —> detection of antigenic properties of bacteria by serotyping

• Phage-typing —> susceptibilty to bacteriophages

• Physiochemical —> detection od physiochemical content of cells by spectrometry

• Molecular Genetic —> detection of specific nucleic acid sequence of bacteria

Microscopical Evaluation

• shape + arrangement of cells

• gram staining properties

• acid fastness

• spore formation

• Exchericia coli

• Klebsellia pneumoniae

• Proteus mirabilis

• Salmonella spp

• Haemophilus influenza

• Staphylococcus auris

Proteus mirabilis

is for the detection of cytochrome c oxidase

• cytochrome c oxidase can remove electron from compound to O2 of encironment

• if cytochrome c oxidase is present it oxidizes the indicator TPMD —> color of indicator changes to blue

POSITIVE —> Pseudomonas aeruginosa

NEGATIVE —> E. Coli

catalase protects bacteria from toxic properties of hydrogen peroxide —> if bubbles are formed the bacterium has catalase —> catalase test is positive

POSITIVE:

• Staphylococcy (eg: aureus)

NEGATIVE:

• Streptococcy (eg: agalactiae)

• Enteroccocy

differentiation between aerobic + facultative anaerobic bacteria —> metabolizing of glucose in aerobic + anaerobic environment

• bacteria metabolises glucose (green —> medium turns yellow (aerobic) —> oxidative metabolism

• if the medium stays green —> negative

• if medium turns yellow after we add parafine oil —> fermentative metabolism = anaorobic

E. Coli

• anaerobic + aerobic positive

• oxidation + fermentation metabolism

Pseudomonas aeruginosa

• aerobic —> positive —> oxidative metabolism

• anaerobic —> negative —> NON-fermentive metabolism

utilization of glucose is connected with production of acids, thus pH in medium is lowered and indicator changes color —> produced gas will accumulate in the small inverted tube —> bubble will form

E. Coli —> Positive

Shigella spp. —> Negative

Motility Test: Inoculation of bacterium to semiliquid mediuma —> motility of bacteria at inoculated line —>mist

Indole Test: tryptophana —> indole indicator —> contains aldehyde (eg: Kovacs reagent) —> red ring

Urease Production Test: urea —> ammonia, H2O, CO2

• alkalizing of pH, changing the color of pH indicator (fenolftalein)àpurple color

E. Coli

• Motility: +

• Indole formation: +

• Urea breakdown: -

• lactose: +

• gas: +

Proteus mirabilis:

• M: +

• I: -

• U: +

• lactose: -

• hydrogensulfate: +

Klebsellia pneumoniae:

• M: -

• I: -

• U: +

• Lactose: +

• gas: +

DNase is extracellular enzyme that cleaves DNA to oligonucleotidesa —> it has leucocidal property

Principle: DNA included in medium is cleaved by DNase produced by bacterium —> production of DNase is visualized by overlying medium with HCl —> DNA is denaturized and transparent zone is visible in positive result

POSITIVE: Staphylococcus aureus (black halo —> no clot formation)

NEGATIVE: Staphylococcus epidermidis (white halo —> whiet halo)

• direct microbiological method —> according to the antigenic structure of bacteria

• reaction of antigens of bacterium with known sera (detecting antigenic properties) —> result —>

  serotype

• Importance: identification of bacteria or epidemiological reasons —> Samonella spp., Neisseria meningitidis, Haemophilus influenzae

• antigenic structures serve as specific receptors for bacteriophages

• succeptibility of bacteria to various phages reflects its antigenic structure —> determination of phagotype of bacterium

• for identification + epidemiological characteristics of bacterial strain

• Mass Spectrometry is method that allows to analyze components of specimen according to their molecular weight and charge

• MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) —> is one of the modifications of mass spectrometryàanalyzes proteins of microbial cells —> used for rapid + exact identification of broad spectrum of microbes

  
  

• bacterial strains in pure culture of specimen containing single strain are required

• limitations of MALDI-TOF MS —> cannot reliably distinguished eg E. Coli + Shigella ssp. —> better biochemical test or generally identification according to the specific nucleic acid sequences

MALDI-TOF MS

1. Ionizing of molecules of specimen

2. ionized molecules fly through tube and separated according to their molecular weight + electric

   charge

3. they achieve detector and mass spectra are producedàratio of molecular weight and charge

4. spectra are compared with database and bacteria is identified

These methods allow rapid + exact identification of bacteria by analyzing of their specific nucleic acid —>from pure culture and directly from biological specimen too.

• -  Hybridization —>probes

• -  Hybridization —> amplification —>PCR

• -  Sequencing