Chapter 20: Microorganisms Encountered in the Respiratory  Loeffler’s agar slant: starvations medium which enhances

Tract pleomorphism of C. diphtheria

UPPER RESPIRATORY TRACT
*sore throat is the most common infection in the upper respiratory tract
1. Culture of Specimen from URT would
 Aids in the diagnosis of certain infections [sore throat, diphtheria,
candidiasis (thrush)]
 Establish the focus of infection in certain diseases (scarlet fever,
rheumatic fever, acute glomerulonephritis)
 Detect carrier state of organism (group A beta-hemolytic streptococci,
meningcocci, Staphylococcus aureus diphtheria bacilli)
*best results are obtained when specimen is taken before antimicrobial
therapy and specimen should be taken properly
 Cystine Tellurite agar: differentiates the 3 types of C.
diphtheriae colonies
 In suspected cases of C. diphtheriae, make an additional
smear of the specimen to be stained with Loeffler’s
methylene blue to demonstrate the metachromatic
granules
 Bordet – Gengou medium: medium of choice for isolation of
B. pertussis
 Regan – Lowe / Charcoal Horse BA – more sensitive than
Bordet – Gengou for B. pertussis
 Modified Thayer – Martin or Martin – Lewis: for isolation of
N. meningitidis (detection of carriers) and N. gonorrhoeae;
incubated in 5 – 10% CO2 for 48 hours
LOWER RESPIRATORY TRACT
1. Lower Respiratory Tract Disease

2. Collection of Specimens  Acute Bronchitis

 Chronic Bronchitis
*cotton and Dacron or calcium alginate tipped swabs (last two: isolation of  Pneumonia (2 major causes)
group A beta-hemolytic strep & will also prolong the viability)  Community acquired
 Common etiologic agents
 Throat swab  Children (2 months – 5 years old)
 Group A beta-hemolytic streptococci  RSV (respiratory syncytial virus)
 Cornybacterium diphtheriae  Parainfluenza virus
 Mycoplasma  Influenza virus
 Chlamydia  Adenoviruses
 Haemophilus species  Neonates: C. trachomatis or Pneurocystis carinli
 Yeast (oral thrush/candidiasis)  Young adults (< 30 years old): M. pneumonia
 Take specimen from areas with exudate; avoid areas with  Adults suffer less bacterial pneumonia caused by H.
normal flora influenzae, S. pneumonia and S. aureus
 Nasopharyngeal swab  Hospital acquired
 B. pertussis  Klebsiella sp. And other Enterobacteriaceae, S. aureus,
 Neisseria sp. anaerobes, S. pneumonia, P. aeruginosa, Legionella
 Cornybacterium diphtheriae  Chronic LRT Infection
 Use platinum/aluminium wire loop  M. tuberculosis: most common
 Aspirated Nasopharyngeal Secretions  MOTT: M. avium – intracellulare and M. kansasii
 Best specimen for B. pertussis  Actinomyces and Nocardia
 Ideally inoculated directly to fresh culture media at patient’s
bedside (if not directly inoculated, transport for not < 2 hours in
2. Specimens
Aimes TM with charcoal)
 Nasal swab
 Anterior nares cultures are occasionally required for the study of  Expectorated Sputum
staphylococcal carriers  For pxs who cooperate
 Most common and is highly contaminated with URT secretions
 First early morning specimen: best specimen for most
3. Transport
purposes since it represents a pool of material accumulated
overnight
 After collection, swabs must remain moist and inoculated immediately  Gastric aspirate: may be collected from patients who are
within 4 hour but if there is a longer delay, you may refrigerate for 2-3 unable to produce sputum
hours and held at RT within 24 hours
 Exclusively used for the isolations of AFB
 Stuart’s TM or Amies TM (prolong the viability of the organism)
 Other Specimens Obtained from the LRT Without Oropharyngeal
Contamination
4. Laboratory Diagnosis  Bronchoscopic specimen
 Tracheostomy and endotracheal aspirates
 Microscopic Examination  Transtracheal or infralaryngeal aspirates
 Gram stain: to determine what possible organisms are present in  Lung aspirates
the specimen (by checking the morphology)  Lung biopsies (needle aspirations)
 To diagnose Vincent’s angina and oral thrush *all of these except for bronchoscopy are for AFB isolation
 Vincent’s angina - exudative pharyngitis brought by  In Legionella pneumonia, sputum may be scanty and watery, with
certain anaerobic oral flora (F. necrophorum – most few or no host cells. Such specimens may be positive by direct
commonly isolated); characterized by a membrane type florescent antibody stain and culture, and they should not be
of lesion similar to diphtheria but accompany by foul subjected to screening procedures
odor (characteristic fusiform bacteria & spirochete will  Sputum from patients with CF should be screened. A throat swab
be seen) is an acceptable specimen from patients with CF in selected
 10% KOH: to visualize fungal elements including yeast cells and clinical settings and should be processed in a similar manner as CF
pseudohyphae (indication of fungal infection) sputum

 Culture 3. Transport
 Routine culture: 5% sheep BA (CO2), MacConkey, CA(CO2)
 Multiple interrupted streaking with heating  Specimen should be examined without delay or transported within 2
 Additional media may be used: hours
 SSA (Streptococcal Selective Agar): will supress the normal  Refrigeration of specimen for not > 24 hours (2-3 hours) is satisfactory
flora and other beta-hemolytic streptococci except Group A for the recovery of most pathogens
and B; this will hasten the isolation and ID of organisms
4. Laboratory Examination
 First observe macroscopically (describe the gross appearance)
 Microscopic examination
 Gram stain
 Test of reliability
 Will evaluate the quality of expectorated sputum received for
routine bacterial culture
 Purulent culture is carefully selected  glass slide  air dry
 stain  LPO (100x)
 Slide should be scanned 100x and the number of
squamous epithelial cells (SEC) and
polymorphonuclear (PMN) leukocyte noted
 Ideal specimen: < 10 SEC, > PMN/LPF
 Acceptable specimen: < 10 SEC/LPF
 Exception: patients who are neutropenic
 Contaminated specimen: > 10 SRC/LPF
 Acid fast stain: classic Ziehl – Neelsen or Kinyoun carbol fuchsin
 2 smears each for direct and concentration method
 Culture
 Endotracheal aspirates (ETAs) from mechanically ventilated adult
patients can be screened by gram stain
 Criteria used to reject ETAs from adult patients included greater
than 10 squamous epithelial cells per LPO field or no organisms
seen under OIO
 Exception: in Legionella pneumonia, sputum may be scanty
and watery, with few or no host cells. Such specimens may
be positive by direct florescent antibody stain and culture,
and they should not be subjected to screening procedures
 Sputum from patients with CF (cystic fibrosis) should be screened.
A throat swab is an acceptable specimen from patients with CF in
selected clinical settings and should be processed in a similar
manner as CF sputum
 For routine culture: 1-3mL of sputum is needed
 5% sheep BA (CO2), CA (CO2), MacConkey, Thio
 Pick out purulent or bloody portions of the specimen for
inoculation
 For increased recovery of Hi, incorporate bacitracin into CA
 AFB culture
 Ziehl – Neelsen or the Kinyuon carbol fuchsin stain
 Auramine or auramine – rhodamine can also aid in detecting
AFB (more sensitive and more expensive)
 10mL of sputum
 2 LJ slants
 Any typical growth of possible pathogen  do ID
 Method of streaking (CVGH) for routine culture: multiple
interrupted streaking with heating in between areas of streaking
 Purpose: quantitation (BA)
 Organisms causing inflammation will be present in greater
numbers than any other superficial contaminating organism
 Semiquantitation:
Colony Numbers Growth Density
1 -24 Very scanty
25 - 100 Scanty
Numerous, but discrete Moderate
Confluent, at least within Heavy
the inoculum well
Chapter 21: Microorganisms Encountered in the CSF
- No indigenous flora (sterile body fluid)
- Bacteriologic examination of spinal fluid is an essential step in the
diagnosis of any case of suspected meningitis
- Bacterial meningitis
o 95% of cases: children < 5 years old
o Newborns (different bacteria from other age groups
because they come from the vaginal hole of the mother)
 Group A beta-hemolytic streptococci
 E. coli
 Other gram (-) bacilli
 L. monocytogenes
o Children (between 1 mo. – 6yrs. old): H. influenza type
B (most common but has now been reduced because of
newborn screening)
o Children (6 years old) : meningococcal or
pneumococcal meningitis
o Can be diagnosed through microscopic examinations,
chemical studies and hematology
o CSF: purulent
 Numbers of inflammatory cells (PMN) >
1000/ cumm – neutrophils
 Glucose levels < 45 mg/dL
 Protein concentration > 100 mg/dL
o May be acute: usually caused by the encapsulated
bacterial species (ex. H. influenzae & S. pneumoniae)
o May be chronic: caused by M. tuberculosis, other
bacteria, fungi (patient would have tuberculous
meningitis)
- Tuberculosis meningitidis
o CSF is non purulent
o Reduced glucose content
o CHON increase (> 50 mg/dL)
o Low white cell count of mononuclear type
1. Collection, Transport and Initial Handling of Specimen
 Lumbar tap
 Skin must first be decontaminated to avoid normal flora
 Hand delivered immediately to the laboratory (because most are
fastidious organisms)
 Should NEVER be refrigerated
 If not processed immediately, incubate at 37C or leave at RT for 6
hours
 If specimen is suspected of hatbouring a virus, refrigerate for up to
24 hours after collection or frozen at -7-C
 Concentrated by centrifugation
 Sediment – Microbiological studies
 Supernatant – Chemical studies
 3 or 4 tubes should be collected
 Tube 1 & 2: Microbiological and chemical studies
 Tube 3 & 4: Cell count and differential
 We use the last two tubes because usually when the
doctor collects the first two tubes its still clear while the
last two tubes contain blood
 Volume: 1-5 mL
2. Laboratory Examination
 Microscopic Examinations
 Gram stain, Acid fast stain, India ink preparation
 Acridine orange flourochorme stain may allow faster examination
of stain under HPO; bacteria will flouresce
 Slides must be very clean to avoid false (+) smears (use autoclave
slides)
 To make smears for staining (gram and AFB)
 Centrifuge and use the sediments
 A heaped drop of a well – sedimented specimen is
placed on the surface of sterile slide (use a Pasteur
pipette)
 A second heaped drop is applied in the same are after
the first drop has dried (optional)
 Purpose: to increase the concentration of the material
 Sediments: should NEVER be spread out on the slide
surface because you will have difficulty in finding
mircoorganisms
 India ink preparation
 Visualization of Cryptococcus neoformans due to their
large polysaccharide capsule
  Presence of encapsulated buds, smaller than mother cell
is diagnostic
 Culture
 Several drops of the sediment should be inoculated to each
medium
 Method of inoculation: multiple interrupted streaking WITHOUT
heating in between areas of streaking (because specimen is already
sterile)
 5% sheep BA (CO2), BA (aerobically); CA (CO2); Thio
(enrichment broth); MacConkey
 Plates are incubated for 18 – 24 hours (max of 72 hours) at 37C
 Broth is incubated at 37C aerobically for 5 days
 Any growth of possible pathogens: do ID
Chapter 21: Sterile Body Fluids
- Clear fluids: concentrate by centrifugation or filtration
- Purulent fluids: inoculate directly to the media
- Clotted fluids
o Homogenize to release trapped cells
 Tissue homogenizer
 Sterile mortar and pestle
 Glass tissue grinder
o Mince/cut to release fungal cells
1. Pleural Fluid
 Types
 Transudate – may contain few or no cells and be a consistency
similar to serum but with low protein content
 Exudate – numerous WBC and other evidence of inflammatory
response usually due to infection
 Thoracentesis (method of ollection)
 Submitted as pleural, thoracentesis or empyema fluid (exudative
pleural fluid that contains numerous PMN’s neutrophils and are
grossly purulent)
 1-5 mL
 Use sediments for smear or culture
2. Peritoneal Fluid
 Normal fluid: contains as many as 300 WBC/mL
 Protein and specific gravity are quite low
 During infections or inflammatory process, increased amounts of
fluid accumulate in the peritoneal cavity ascites or ascitic fluid
(contains an increased number of PMNs and elevated protein level)
 Paracentesis (method of collection)
 1-5 mL
 Use sediments for staining and culture
3. Pericardial Fluid
 The area between the heart muscle and pericardium, pericardial space,
normally contains 15 – 20 mL clear fluid
 Pericarditis - inflammation of pericardium, usually viral
 Myocarditis – inflammation of the heart muscle
4. Joint Fluid
 Synovial fluid
 Arthritis
 Arthrocentesis (method of collection)
5. Etiologic Agents
 S. pneumoniae
 S. aureus
 Enterics
 Other gram (-) bacilli
 Anaerobes
 Group A beta-hemolytic streptococci and other streptococci
 Fungi, viruses and occasionally parasites
Chapter 23: Wound infections, Abscesses, Ulcers
 S. aureus; S. pyogenes; E.coli; Bacteriodes; Fusobacterium; Proteus’
Moragnella, Providencia; other enterobacteriaceae; Pseudomonas sp.,
Clostridium sp.; Peptostreptococcus; microaerophilic streptococci;
nonsporeforming anaerobic gram (+) rods; Candida (mostly obliquely
anaerobes)
 How to isolate under anerobic conditions
 Inoculate to Thio and CMM (cooked meat medium) – other
organisms can grow on this medium but it is especially for
anerobes
 Incubate then smear (gram stain), you will observe gram (+)
sporeforming rods
 Heat shock using CMM and do this for 30 minutes
 Purpose: To kill the non sporing bacteria
 Subculture on blood agar
 Gas Pak jar (for anaerobes) - (5-10% CO2; 5-10% H2; 80 – 90% N2)
 Utilize a clear, heavy plastic with an airtight lid that is clamped
down to prevent leakage
 Introduction of a gas mixture containing H2 into the jar is followed
by catalytic conversion of O2 in the jar with H2 to H2O thus
establishing anaerobiosis
 Catalyst: palladium coated alumina pellets
 Reactivation or rejuvenation of catalyst: 160C for 1.5 – 2 hrs
 Generator envelope: sodium borohydrate & citric-acid-sodium
bicarbonate (also establishes anaerobiosis)
 Indicator: methylene blue or resazurin (placed inside the jar)
 Open envelopes is placed in a jar, water is added & the jar is
sealed
 Incubate for 48 hours at 37C
 Production of heat within a few minutes & subsequent
development of moisture on the inner walls of the jar are
indications that the catalyst & generator envelope are functioning
properly
Chapter 24: Tissue
 Surgery/needle biopsy or autopsy
 Place in a wide mouthed, screw capped bottle or plastic container
without any fixative (for microbiological studies; fixative kills bacteria)
 In case of contaminated material:
 To reduce surface contamination of the material:
 May be surface cauterized with an electric soldering iron or
heated spatula
 Blanched by immersing in boiling water for 5 – 10 seconds
 Specimen may be then dissected with sterile
instruments to permit culturing the center of the
specimen not affected by heat
 Culture media will depend largely on what organisms are being sought
or suspected
 Preparation of tissue for culture
 Finely minced with sterile scissors and transferred to tissue grinder
 Grind to pasty consistency using sterile sand or broth
 Tissue and sand are allowed to settle; supernate is transferred to
another sterile tube and used for culture
 Autopsy or post-mortem cultures
 Blood, tissue and aspirates
Chapter 25: Eye Infections
 Due to constant washing activity of tears which have antibacterial
constituents, recovery of organisms in many eye infections may be
relatively low
 *Conjunctivitis,* keratitis, endophthalmitis, periocular infections (*most
common)
 Etiologic agents that may be recovered from bacterial conjunctivitis:
 S. pneumoniae, S. aureus, H. influenza, C. trachomatis, viruses,
fungi, parasites, Pseudomonas aeruginosa
 Conjunctiva: used aerobic swab moistened with Stuart’s or Amies TM
(premoistened with sterile saline)
 Transport within 24/RT
 Direct visual examination
 Gram stain and/or other appropriate techniques
 Bacterial conjunctivitis: PMN leukocytes predominate
 Viral conjunctivitis: lymphocytes and monocytes
predominate
 Herpes infection: multinucleated epithelial cells; intracellular
inclusion bodies (Tzanch – Wright – Giemsa)
 Chlamydia: basophilic intracytoplasmic inclusion bodies in
epithelial cells (Giemsa)
 Fungi: 10% KOH
 Cultivation
 Eye: BA, CA(CO2), BA(anaerobic), Thio
 Selective media may be used
 M. lacunata – Loeffler’s medium
 C. diphtheriae (diphtheric conjunctivitis) – Loeffler’s/Cystine
Tellurite agar

Chapter 26: Ear Infections

 Middle and inner ear are usually sterile

 Tympanocentesis – otolaryngologist
 External ear usually reflect the microbiota of the skin; therefore, diseases
of the outer ear will reflect disorders of the skin
 Outer ear – aerobic swab moistened Stuart’s or Amies TM and
transported within 24 hours/RT
 Inner ear – sterile screw cap tube or anaerobic transporter and
transported immediately to lab at RT
 Storage: 6 hrs at RT
 Acute/Subacute Otitis media
 Pyogenic cocci: S. pneumoniae, S. pyogenes, S. aureus
 H. influenzae
 Chronic Otitis media
 Psuedomonas, anaerobes, enteric bacilli (Proteus spp.)
 Cultivation
 BA, CA(CO2), Thio, BA (anaero), PEA [selective medium for
gram (+)]