A1 Methods to identify bacteria

- 2 methods:
o Bacteriologic: staining and culturing the organism
o Immunologic: detecting antibodies against organism in the patient’s serum
- 3 methods of bacteriologic lab work:
o Observing under microscope after staining
o Obtaining pure culture via inoculation onto bacteriologic medium
o Identifying organism using biochemical reactions, growth culture, dna probes…
- Blood agar – RBCs with no nucleus – obligate intracellular bacteria and viruses cant grow –
e.g chlamydia and rickettsia
- Steps in bacterial diagnosis
1. Obtain specimen from infection site
2. Stain specimen – gram stain or acid fast stain – genus may be found
3. Culture the specimen on media such as blood agar – obtaining isolated colonies
(pure culture is important)/ incubate plates with presence or absence of oxygen
4. Identify organism – special tests – dna probes, dna fermentation, antibody
5. Perform antibiotic susceptibility tests
- Neisseria and haemophilus are grown on chocolate agar (heated blood agar) because the
blood contains inhibitors which must be inactivated through heating
- Blood cultures:
o Organisms frequently identified through this are: two gram-positive cocci,
Staphylococcus
aureus and Streptococcus pneumoniae, and three gramnegative rods, Escherichia coli,
Klebsiella pneumoniae, and
Pseudomonas aeruginosa
- Throat cultures:
o primarily to detect the presence of
group A -hemolytic streptococci (Streptococcus pyogenes),
an important and treatable cause of pharyngitis.
o also used when diphtheria, gonococcal pharyngitis, or
thrush (Candida) is suspected
o Gram stain is typically not done on a throat
swab because it is impossible to distinguish between the
appearance of the normal flora streptococci and
S. pyogenes
- Sputum culture:
o Pneumonia or tuberculosis are suspected
o most frequent cause of
community-acquired pneumonia is S. pneumoniae, whereas
S. aureus and gram-negative rods, such as K. pneumoniae
and P. aeruginosa, are common causes of hospital-acquired
pneumonias
o If Legionella pneumonia is suspected,
the organism can be cultured on charcoal-yeast agar, which
contains the high concentrations of iron and sulfur required
for growth
o If tuberculosis is suspected, an acid-fast stain should be
done
- spinal fluid culture:
o performed primarily when meningitis is suspected.
 o important causes of acute bacterial meningitis are three encapsulated organisms:
Neisseria
meningitidis, S. pneumoniae, and Haemophilus influenzae
o the quellung test or immunofluorescence with
specific antisera can identify the organism rapidly
o subacute meningitis, Mycobacterium tuberculosis and the fungus Cryptococcus
neoformans are the most
common organisms isolated  Acid-fast stains
o C. neoformans,  india ink
- Stool cultures:
o primarily for cases of enterocolitis.
o Shigella, Salmonella, and
Campylobacter. E. coli  common causes of diarrhoea
o Gram stain may reveal large numbers of certain organisms,
such as staphylococci, clostridia, or campylobacters. Gram
stain of the stool is not usually done because the large numbers of bacteria in the
normal flora of the colon make the
interpretation difficult
o Salmonella and Shigella, a selective, differential medium such as MacConkey or Eosin–
Methylene
Blue (EMB) agar is used
- Urine cultures:
o pyelonephritis or cystitis is suspected
o most frequent cause of
urinary tract infections is E. coli. Other common agents are
Enterobacter, Proteus, and Enterococcus faecalis
- genital tract cultures:
o from individuals with an abnormal discharge or on
specimens from asymptomatic contacts of a person with a
sexually transmitted disease
o pathogens in the genital tract is Neisseria gonorrhoeae
o swabbing the urethral canal
(for men), the cervix (for women), or the anal canal
o Chlamydia trachomatis,
- Bacteriologic agar types:
o Blood: detects hemolysis
o Charcoal yeast extract : legionella pneumophilia – increased conc of iron and
cysteine
o Chocolate: Neisseria meningitidis and gonorrhoea – heated blood inactivates
inhibitors of growth
o Eosin methylene blue: enteric gram negative rods – against gram positive bacteria,
differentiates bwteen lactose fermenters/ non fermenters
o Macconkey: same as eosin meth. Blue
- 5 laboratory techniques:
1. Microsocpic visulaisation
2. Cultivation and identification
3. Detecting microbial agents
4. Detecting microbial dna or rna
5. Detecting inflammatory or host response to microorganism
- Take a patiet history and physical examination:
o Example: cough  respiratory tract infection; dysuria  urinary tract infection
o Age of patient
o Where they have been
o Where they work
o Example: gram + coccus in spinal fluid of new born likely to be strep. Agalactiae
rather than strep. Pneumoniae
o Example: Erythema migrans (skin lesion, bright red) indicates lyme disease
- Direct visualisation of the organism:
o Gram stain, acid-fast stain, india ink preparation, potassium hydroxide,
o Microbes excluding viruses can be visualised microscopically
o Gram stain
1. Heat fix specimen to slide, add crystal violet wait 1 minute
2. Rinse slide, add iodine, wait 1 minute, purple organisms indicate gram
+
3. Rinse, decolorise with acetone for 5 seconds
4. Was slidewith water, gram – aren’t visible
5. Apply safranin counterstain
6. Wash in water, dry in air, gram – are visualised as red
 Mycoplasma have no cell wall so cannot be visulaised through gram
staining
 Visualisation requires > 10 to power of 4 organisms/mL of liquid, low conc.
Sample are centrifuged
o Acid fast stain= ziehl neelsen (classic), to identify mycolic acid in organisms cell
wall, mycobacterium tuberculosis which appears pink, beaded, curved, for
patients with mycobacterial infections
o India ink = to detect cryptococcus neoformans in CSF
- Growing bacteria in culture:
o Used mainly for bctria or fungi not for helminths or protozoa
o Example = streak a throat swab onto blood agar to search for group A beta
hemolytic streptococcus
o Use characteristics such as colony, size, shape, colour, gram stain, hemolysis, odor,
metabolic properties
o Pure cultrues = used for antimicrobial susceptibility testing
o Specimen collection:
 Transport in proper conditions to prevent inadequate samples
o Growth requirememtns:
 Most bacteria are heterotrophs = require organic carbon for growth
 Fastidious = require large number of growth factors or nutrients and must
be specific for them
o Oxygen requirements:
 Aerobes = need oxygen, oxidative phosphorylation
 Anaerobes = fermentation, no oxygen
 Facultative anaerobes = grow in absence of o2 but grow better in its
presence
 Aerotolerant anaerobes = protects itself from oxygen but don’t use it in
their metabolism
 Microaerophiles = need o2, cannot survive at atmospheric levels of
oxygen, found in lakes or wet soil
o Media:
 2 strategies to isolate bacteria
 enriched media = non selecyive growth of any bacteria
 Selective media = growth of specific bacteria from specimens with
large numbers of bacteria
 Enriched media
 Media containing: blood, yeast extract, brain/heart infusions for
growing fatsiious organisms
 Sheep blood agar = protein, sodium chloride, sheep blood,
supports most gram +/- bacteria
 Chocolate agar = RBCs that have been lysed through heating,
releases Hb, hemin (X factor), NAD for organisms such as
haemophilus influenzae and Neisseria gonorrhoeae
 Useful to culture blood and csf which are normally sterile
 Selective media
 Macconkey agar =
o Promotes growth of gram – rods, such as
enterobacteriaecae;
o inhibits growth of gram + organisms and some fastidious
gram – bacteria (haemophilus and Neisseria)
o used to detect organisms able to metabolise lactose
 hektoen enteric agar
o lactose/sucrose fermenters + non fermenters
o h2s producers
o Culture: salmonella and shigella species
 Thayar martin agar
o Isolate gonococci
o Identifying bacteria
 Single enzyme tests:
 Catalase: H202  h20 + o2 ; organisms produce bubbles if catalase
+; differentiates between staph. (catalase +) and strept./
enterococci (catalse -) ; it is a virulence factor because h202 is
antimicrobial so it shows how well organisms can survive against
drugs
 Oxidase: differentiaties gram – bacteria, pseudomonas aeruginosa
is oxidase positive, cytochrome c oxidase (ETC, nitrate metabolism)
accepts substrates such as phenylenediamine derivatives
producing a dark product in bacteria
  Urease: urea + h20  2nh3 + co2 urease hydrolyses urea to
ammonia, pH test identifies ammonia through colour change,
identifies enterobacteriacae such as Corynebacterium urealyticum,
helicobacter pylori
 Coagulase: enzyme causes clot to form when bacteria are
incubated with plasma, differneittates staph. Aureus (coag. +) from
staphylococci (coag. -)
 Automated systems:
 Vitek system: using small plastic reagent cards, data analysed by
computer, simultaneous identification and antimicrobial
susceptibility testing
o Immunologic detection
 Detection of antigen with known antiserum
 Quelling reaction: to visualise capsules more clearly; s.
pneumoniae, h. influenzae b, Neisseria meningitidis A+C
 Slide agglutination test: salmonella + shigella are agglutinated
using specific antibodies  cross linking
 Identifying serum antibodies
 Complement fixation:
o Ability of antibody to bind complement,
o First add antigen then complement proteins
o Patients serum with IgG or IgM antibodies forms an
antibody-antigen-complement complex  little
complement remains
o Then antibody coated rbcs are added
o The rbcs wont lyse if less complement is available
o Monitor the test with spectrophotometer  lysed rbcs
release Hb
 Direct agglutination:
o Used for evaluating presence of brucella abortus or
francisella tularensis
 Direct hemagglutination:
o Rbcs clump when exposed to antibodies in patients serum,
o Mycoplasma pneumoniae
 Other tests:
 Latex agglutination test:
o To test CSF in meningitis
o Latex particles are coated with either antibody or antigen
o Identification of beta hemolytic streptococci group A
 ELISA:
o Antibody bound to microtiter well, patient serum
incubated in wells,
o A second antibody is added with enzymes for a substrate
producing a colour if activated
o Nucleic acid test
 Non amplified assay (direct hybridisation)
  Detect short sequence of nucleotide bases of DNA unique to
pathogen
 Hybridisation with a probe
 Common targets are the 16S rRNA gene as it has multiple copies
 Amplification using PCR
 PCR: amplification with dna polymerase of unique sequences
 Gel electrophoresis
 Real-time PCR: fluorescent,
 No need to culture microorganisms, just get target sequences and
amplify them many time over
 DNA microarrays:
 Used to measure gene expression
 can detect and identify many pathogens from same specimen
 dna oligonucleotides (probes) are used
 fluorescence probes are used
o susceptibility testing
 disk diffusion method:
 the disk contains exact amounts of antimicrobial agents
 it is added to inoculated culture dishes with the microorganism to
be tested
 zone of growth of inhibition is measured
 minimal inhibitory concentration
 used to detect the minimal amount of antibiotic needed to kill the
bacteria
 using different dilutions of the antibiotic
 lowest concentration of antibiotic that kills 100% of bacteria
 bactericidal vs bacteriostatic drugs:
 statsic arrest growth and replication
 icidal kills bacteria