COMPREHENSIVE REVIEW PARTS 2 AND 3

MICROBIOLOGY AND PARASITOLOGY

1. Staphylococci and Micrococci

Differentiation of Staphylococcus from Micrococcus
Staphylococcus Micrococcus
Catalase + +
Aerobic growth + +
Anaerobic growth + -
Glucose utilization (OF media) Fermentative Oxidative or nonsaccharolytic
Modified oxidase - +
Benzidine - +
Resistant to lysostaphin (200 g/ml) - +
(susceptible) (resistant)
Resistant to bacitracin (0.04 U) + -

Staphylococcus Gram-stain: gram-positive cocci arranged in Bound coagulase (clumping factor): positive
aureus clusters Free coagulase: positive
Medium to large, raised colonies on blood agar MSA: growth and fermentation
and CNA with cream to golden yellow DNAse: positive
pigmentation Thermostable nuclease: positive
Beta haemolytic or nonhemolytic on sheep blood
agar
Staphylococcus Gram-positive cocci in clusters To differentiate from S. aureus: S. epidermidis
epidermidis White, creamy, raised growth on blood agar is coagulase negative, DNase negative and
Nonhemolytic on blood agar cannot ferment mannitol.
Growth, but lack of fermentation on MSA To differentiate from S. saprophyticus: S.
Coagulase negative epidermidis is susceptibe to novobiocin; S.
DNase negative saprophyticus is resistant to novobiocin.
Susceptible to novobiocin

2. Streptococci
Streptococcus species
Gram-positive cocci occurring in pairs or Some species show enhanced growth
chains under increased CO2
Nonmotile Requires supportive or enriched media
Non-sporeforming such as blood agar
Facultative anaerobes Susceptible to vancomycin
Catalase negative

Group A strep Infections include pharyngitis, Beta-hemolytic PYRase positive

Streptococcus tonsillitis, or strep throat
pyogenes scarlet fever, impetigo,
cellulitis, wound infections
and erysipelas
May be associated with acute
rheumatic fever (ARF)
Acute glomerular nephritis
(AGN) may be seen in
patient following
streptococcal pharyngitis or
skin infections
Group B strep Associated with neonatal
Streptococcus infections, including
agalactiae pneumonia, meningitis and
bacteremia
Groups C, F and G Causes severe pharyngitis
strep followed by bacteremia
Oppurtunistic pathogen that
may also be associated
with pneumonia, cellulitis,
and abscess
Group D UTI and wound infections
Enterococcus
Group D
nonEnterococcus
Susceptible to bacitracin
Resistant to trimethoprim
sulfamethoxazole
Beta-hemolytic CAMP positive
Resistant to bacitracin
Resistant to trimethoprim
sulfamethoxazole
Beta-hemolytic A small percentage of groups
Resistant to bacitracin C, F and G are susceptible
Susceptible to trimethoprim to bacitracin
sulfamethoxazole
Alpha, beta or nonhemolytic Bile esculin positive
Resistant to bacitracin Growth in 6.5% salt broth
Resistant to trimethoprim PYRase positive
sulfamethoxazole
Alpha, beta or non-hemolytic Bile esculin positive
Resistant to bacitracin No growth in 6.5% salt broth
Resistant to trimethoprim PYRase negative
sulfamethoxazole
 Page |2

Streptococcus Lobar pneumonia and Alpha-hemolytic Bile solubility positive

pneumoniae community-acquired Susceptible to optochin Neufeld reaction (Quellung
pneumonia capsular swelling) positive
Viridans streptococci Most are normal flora of the Alpha-hemolytic Bile solubility negative
human oropharynx and are Resistant to optochin
important agents of
subacute bacterial
endocarditis

3. In the CAMP reaction, an arrowhead zone of hemolysis forms when group B Streptococus (S. agalactiae) is streaked
perpendicularly to a beta-hemolytic strain of S. aureus. The CAMP factor is an extracellular, thermostable antigenic
protein produced by group B Streptococcus.

4. Occasionally, nonbeta-hemolytic strains of S. agalactiae may be encountered, but identification of such isolates can
be accomplished using the serologic agglutination approach. (Bailey and Scotts)

5. Neisseria
Neisseria Gram-negative diplococcic resembling tiny coffee N. gonorrhoeae requires CAP and N.
species beans or kidney beans except N. elongate that meningitidis and M. catarrhalis require BAP as
is rod-shaped the minimal growth standard. The pathogenic
Obligate aerobes Neisseria grow optimally between 35o and
Capnophilic 37oC.
Cytochrome oxidase positive The Neisseria organisms establish disease
through attachment to mucous membranes of
the host through pili. Pili also participate in
conjugation and transfer of genetic material.

6. Enterobacteriaceae
TSI Reactions
TSI Reactions Typical Organisms
A/AG H2S Glucose with acid and gas Escherichia
Lactose and/or sucrose with acid and Klebsiella
gas Enterobacter
K/AG H2S + Glucose with acid and gas Salmonella
Lactose or sucrose not fermented Proteus
Citrobacter
K/A H2S Glucose with acid; no gas Shigella
Lactose or sucrose not fermented Providencia
Serratia
Anaerogenic Escherichia coli
K/K H2S Glucose not fermented Pseudomonas
Lactose or sucrose not fermented Alcaligenes

Escherichia coli TSI: A/AG Deaminase (phenylalanine): I M V C

H2S: negative negative Urease: negative E. coli + + - -
MacConkey: pink-red colonies LDC: positive
Indole: positive ODC: most strains positive
MR: positive ADH: most strains negative
VP: negative ONPG: positive
Citrate: negative
Motility: positive
Klebsiella TSI: A/AG Motility: negative I M V C
pneumoniae H2S: negative Deaminase (phenylalanine): K. pneumoniae - - + +
MacConkey: pink, mucoid negative K. oxytoca + V + +
colonies LDC: positive K. ozaenae - + - V
Gas: positive ODC: negative
MR: negative ADH: negative
VP: positive Urease: weakly positive
Citrate: positive
Enterobacter TSI: A/AG Citrate: positive Motility: I M V C
species H2S: negative positive E. aerogenes - - + +
Indole: negative Deaminase (phenylalanine): E. cloacae - - + +
MR: negative negative E. agglomerans V V V V
VP: positive ODC: positive
Serratia TSI: K/A VP: positive I M V C
H2S: negative Deaminase (phenylalanine): S. marcescens - V + +
MacConkey: clear colonies negative S. liquefaciens - + + +
Gelatinase: positive LDC: positive
 Page |3

DNase: positive ADH: negative

Lipase: positive Urease: negative
Salmonella TSI: K/AG Indole: negative I M V C
H2S: positive MR: positive Most - + - +
MacConkey: clear (lactose VP: negative Motility: positive serotypes
negative) Deaminase (phenylalanine):
Hektoen enteric: green with negative
black centers (lactose LDC: positive
negative, H2S positive) Urease: negative
Shigella TSI: K/A MR: positive I M V C
H2S: negative Citrate: negative Nonmotile ABC V + - -
Gas in fermentation: negative ODC: negative D - + - -
MacConkey: clear colonies ADH: negative
(lactose negative) Deaminase (phenylalanine):
Hektoen enteric: green colonies negative
(lactose negative) Urease: negative
Proteus TSI: K/AG MR: positive I M V C
H2S: positive LDC: negative P. mirabilis - + V V
Blood agar: growth in waves or ADH: negative P. vulgaris + + - V
swarms Urease: rapidly positive (within P. penneri - + - -
MacConkey: clear colonies 4 hours)
(lactose negative)
Edwardsiella TSI: K/AG Motility: positive I M V C
H2S: positive LDC: positive E. tarda + + - -
Lactose: negative ODC: positive
Indole: positive ADH: negative
MR: positive Deaminase (phenylalanine):
VP: negative negative
Citrate: negative Urease: negative

Differentiation of Proteeae
Reaction P. vulgaris P. mirabilis M. morganii Prov. rettgeri Prov. stuartii
TSI K/AG K/AG K/AG K/A K/A
H2S + + - - -
Gas + + + - -
MR + + + + +
VP - V - - -
Indole + - + + +
Citrate - V - + +
Deaminase + + + + +
(phenylalanine)
Urease + + + + -*
Motility swarms/+ swarms/+ + + +
LDC - - - - -
ADH - - - - -
ODC - + + - -
*Most strains negative

7. Salmonella are positive for lysine decarboxylase and most are negative for KCN. Citrobacter are negative for lysine
decarboxylase and positive for growth in KCN. (BOR)

8. Citrobacter organisms resemble Salmonella but are ONPG positive and LDC negative. (Delost)

9. Morganella is indole positive, VP and citrate negative. PDA and TDA positive. (Ciulla)

10. Providencia species are PDA, TDA, indole and citrate positive and VP negative. (Ciulla)

11. MUG test: Escherichia coli produces the enzyme beta-D-glucuronidase which hydrolyzes beta-D-glucopyranosid-
uronic derivatives to aglycons and D-glucuronic acid. the substrate 4-methylumbelliferyl-beta-D-glucuronide is
impregnated in the disk ang is hydrolyzed by the enzyme to yield the 4-methylumbelliferyl moiety, which fluoresces
blue under long wavelength ultraviolet light.
EXPECTED RESULTS QUALITY CONTROL
Positive Electric blue fluorescence Positive : E. coli
Negative Lack of fluorescence Negative: P. aeruginosa

12. Identification of Pseudomonas aeruginosa

 Page |4

Gram-negative bacilli Indole: negative Motile by one or two flagella

Oxidase: positive Citrate: positive Nitrates are denitrified to nitrites
Pyocyanin: positive LDC: negative Acidamide: positive
Fluorescein (pyoverdin): positive ODC: negative Resistant to kanamycin
Grows well on MacConkey agar ADH: positive Susceptible to carbenicillin
Growth at 42 oC positive Urease: variable

13. A mucoid strain of Pseudomonas aeruginosa has been found to cause severe pneumonia in patients with cystic
fibrosis. Patients with cystic fibrosis activate the alginate gene resulting in the production of alginate, which surrounds
the bacterial cell wall and protects it from phagocytosis. (Delost)

14. Burkholderia cepacia causes nosocomial infections and is also an important respiratory tract pathogen in patients
with cystic fibrosis; second most common cause to P. aeruginosa. (Ciulla)

15. Vibrio species

Gram-negative straight or curved rods Requires increased NaCl except V.cholerae and V. mimicus
Motile with single polar flagellum TCBS agar can be used to isolate most Vibrio species
Facultative anaerobes or aerobic
Oxidase positive

16. Among the vibrios, Vibrio vulnificus causes the most severe disease. Wound infections and septicemia with this
organism are often fatal. Disease is usually associated with consumption of raw oysters or oyster-related injury.
(Henry)

17. HACEK bacteria: Five small gram-negative coccobacilli are part of the normal oral flora and are associated
occasionally with bacterial endocarditis and rarely with other infections. They are opportunists that enter the
bloodstream, settle on damaged heart valves, and cause a relatively slowly progressive, indolent form of endocarditis.
They typically require an additional 12 days before they are isolated from blood cultures, and they are uniformly
susceptible to many antimicrobial agents. The word HACEK is an acronym for the bacteria responsible for this
disease: Haemophilus spp. (influenzae, parainfluenzae), Aggregatibacter (Haemophilus) aphrophilus (most
commonly, Aggregatibacter [Actinobacillus] actinomycetemcomitans), Cardiobacterium hominis, Eikenella corrodens,
and Kingella spp. Some taxonomic changes have been noted more recently, and some of the HACEK members have
been reassigned to the genus Aggregatibacter. (Henry)

18. Plating Media for Routine Bacteriology

MEDIUM COMPONENTS/COMMENTS PRIMARY PURPOSE
Bile esculin agar Nutrient agar base with ferric citrate. Hydrolysis of esculin Differential isolation and
(BEA) by group D streptococci imparts a brown color to the presumptive identification of
medium; sodium desoxycholate inhibits many bacteria group D streptococci and
enterococci
Bile esculin azide Contains azide to inhibit gram-negative bacteria, Selective and differential for
agar with vancomycin to select for resistant gram-positive bacteria, cultivation of vancomycin-
vancomycin and bile esculin to differentiate enterococci from other resistant enterococci from clinical
vancomycin-resistant bacteria that may grow and surveillance specimens
Blood agar Trypticase soy agar, Brucella agar, or beef heart infusion Cultivation of infectious
with 5% sheep blood microorganisms, determination of
hemolytic reactions
Bordet-Gengou agar Potato-glycerol-based medium enriched with 15-20% Isolation of Bordetella pertussis
defibrinated blood. Contaminants inhibited by methicillin
(final concentration of 2.5 g/mL)
Buffered charcoal- Yeast extract, agar, charcoal, and salts supplemented with l- Enrichment for Legionella spp.
yeast extract (BCYE) cysteine, HCl, ferric pyrophosphate, ACES buffer, and -
agar ketoglutarate
Buffered charcoal- BCYE supplemented with polymyxin B, vancomycin and Enrichment and selection for
yeast extract (BCYE) ansamycin, to inhibit gram-negative bacteria, gram-positive Legionella spp.
agar with antibiotics bacteria, and yeast, respectively
Campy-blood agar Contains vancomycin (10 mg/L), trimethoprim (5mg/L), and Selective for Campylobacter spp.
polymixin B (2500U/L), amphotericin B (2 mg/L), and
cephalothin (15 mg/L) in a Brucella agar base with sheep
blood
Campylobacter Thioglycollate broth supplemented with increased agar Selective holding medium for
thioglycollate broth concentration and antibiotics recovery of Campylobacter spp.
Cefoperazone, Blood-supplemented enrichment medium containing Selective medium for isolation of
vancomycin, cefoperazone, vancomycin and amphotericin to inhibit Campylobacter spp.
amphotericin (CVA) growth of most gram-negative bacteria, gram-positive
medium bacteria, and yeast, respectively
Cefsulodin-irgasan- Peptone base with yeast extract, mannitol, and bile salts. Selective for Yersinia spp.; may
novobiocin (CIN) Supplemented with cefsulodin, irgasan and novobiocin; be useful for isolation of
agar neutral red and crystal violet indicators Aeromonas pp.
Chocolate agar Peptone base, enriched with solution of 2% hemoglobin or Cultivation of Haemophilus spp.
isovitalex (BBL) and pathogenic Neisseria spp.
 Page |5

MEDIUM COMPONENTS/COMMENTS PRIMARY PURPOSE

Columbia colistin- Columbia agar base with 10 mg colistin per liter, 15 mg Selective isolation of gram-
nalidixic acid (CNA) nalidixic acid per liter, and 5% sheep blood positive cocci
agar
Cystine-tellurite Infusion agar with 5% sheep blood. Reduction of potassium Isolation of C. diphtheriae
blood agar tellurite by C.diphtheriae produces black colonies
Eosin methylene blue Peptone base with lactose and sucrose. Eosin and Isolation and differentiation of
(EMB) agar (Levine) methylene blue as indicators lactose-fermenting and non-
lactose fermenting enteric bacilli
Gram-negative broth Peptone base broth with glucose and mannitol. Sodium Selective (enrichment) medium
(GN) citrate and sodium desoxycholate act as inhibitory agents for enteric pathogens
Hektoen enteric (HE) Peptone base agar with bile salts, lactose, sucrose and Differential, selective medium for
agar salicin, and ferric ammonium citrate. Indicators include isolation and differentiation of
bromthymol blue and acid fuchsin Salmonella and Shigella
spp.from other gram-negative
enteric bacilli
MacConkey agar Peptone base with lactose. Gram-positive organisms Isolation and differentiation of
inhibited by crystal violet and bile salts. Neutral red as lactose-fermenting and non-
indicator lactose fermenting enteric bacilli

MacConkey sorbitol A modification of MacConkey agar in which lactose has For the selection and
agar been replaced with d-sorbitol as the primary carbohydrate differentiation of E. coli O157:H7
in stool specimens
Mannitol salt agar Peptone base, mannitol, and phenol red indicator. Salt Selective isolation of
concentration of 7.5% inhibits most bacteria staphylococci
New York City (NVC) Peptone agar base with cornstarch, supplemented with Selective for N.gonorrhoeae
agar yeast dialysate, 3% hemoglobin, and horse plasma.
Antibiotic supplement includes vancomycin (2g/mL),
colistin (5.5g/mL), amphotericin B (1.2 g/mL), and
trimethoprim (3 g/mL)
Phenylethyl alcohol Nutrient agar base. Phenylethanol inhibits growth of gram- Selective isolation of gram-
(PEA) agar negative organisms positive cocci and anaerobic
gram-negative bacilli
Regan Lowe Charcoal agar supplemented with horse blood, cephalexin, Enrichment and selective
and amphotericin B medium for isolation of B.
pertussis
Salmonella-Shigella Peptone base with lactose, ferric citrate, and sodium citrate. Selective for Salmonella and
(SS) agar Neutral red as indicator; inhibition of coliforms by brilliant Shigella spp.
green and bile salts
Schaedler agar Peptone and soy protein base agar with yeast extract, Nonselective medium for the
dextrose, and buffers. Addition of hemin, l-cystine, and 5% recovery of anaerobes and
blood enriches for anerobes aerobes
Selenite broth Peptone base broth. Sodium selenite toxic for most Enrichment of isolation of
Enterobacteriaceae Salmonella spp.
Skirrow agar Peptone and soy protein base agar with lysed horse blood. Selective for Campylobacter spp.
Vancomycin inhibits gram-positive organisms; polymyxin B
and trimethoprim inhibit most gram-negative organisms
Streptococcal Contains crystal violet, colistin, and trimethoprim Selective for S. pyogenes and S.
selective agar (SSA) sulfamethoxazole in 5% sheep blood agar base agalactiae
Tetrathionate broth Peptone base broth. Bile salts and sodium thiosulfate inhibit Selective for Salmonella and
gram-positive organisms and Enterobacteriaceae Shigella spp.
Thayer-Martin agar Blood agar base enriched with hemoglobin and supplement Selective for N. gonorrhoeae and
b; contaminating organisms are inhibited by colistin, N. meningitides
nystatin, vancomycin and trimethorprim
Thioglycollate broth Pancreatic digest of casein, soy broth, glucose enrich Supports growth of anaerobes,
growth of most microorganisms aerobes, microaerophilic and
fastidious microorganisms
Thiosulfate citrate- Peptone base agar with yeast extract, bile salts, citrate, Selective and differential for
bile salt sucrose sucrose, ferric citrate, and sodium thiosulfate. Bromthymol Vibrio spp.
(TCBS) agar blue acts as indicator
Todd-Hewitt broth An enrichment broth for streptococci, supplemented with Selective and enrichment for S.
supplemented with nalidixic acid and gentamicin or colistin for greater agalactiae in female genital
antibiotics selectivity thioglycollate and agar reduce redox potential specimens
Trypticase soy broth All-purpose enrichment broth that can support the growth of Enrichment broth used for
many fastidious and nonfastidious bacteria subculturing various bacteria
from primary agar plates

Xylose lysine Yeast extract agar with lysine, xylose, lactose, sucrose, and Isolation and differentiation of
desoxycholate (XLD) ferric ammonium citrate. Sodium desoxycholate inhibits Salmonella and Shigella spp.
agar gram-positive organisms; phenol red as indicator from other gram-negative enteric

MEDIUM
19. Mechanisms of Action for Commonly Used Antibacterial Agents
Antimicrobial Class
Beta-lactams (e.g., penicillin,
ampicillin, mezlocillin,
piperacillin, cefazolin, cefotetan,
ceftriaxone, cefotaxime,
ceftazidime, azotreonam,
imipenem)
Aminoglycosides (e.g., gentamicin,
tobramycin, amikacin, netimicin,
streptomycin, kanamycin)
Glycylglycines (e.g., tigecycline)
Tetracycline
Chloramphenicol
Ketolides (e.g., telithromycin)
Oxazolidinones (e.g., linezolid)
Streptogramins (e.g.,
quinupristin/dalfopristin)
Fluoroquinolones (e.g.,
ciprofloxacin, ofloxacin,
norfloxacin)
Rifampin
Sulfonamides
Trimethoprim
Nitrofurantoin
Lipopeptides (e.g., daptomycin)
Polymyxins (e.g., polymyxin B and
colistin)
Target Sites of Antimicrobial Agents
Cell Wall Synthesis Protein Synthesis
Beta-lactams: penicillins, Aminoglycosides:
cephalosporins, gentamicin, tobramycin,
carbapenems, kanamycin
monobactams Tetracyclines
COMPONENTS/COMMENTS
Source: Bailey and Scotts Diagnostic Microbiology
Mechanism of Action
Inhibit cell wall synthesis by binding
enzymes involved in peptidoglycan
production (i.e., penicillin-binding
proteins, or PBPs)
Inhibit protein synthesis by binding to
30S ribosomal subunit
Inhibition of protein synthesis by
binding to 30S ribosomal subunit
Inhibits protein synthesis by binding
30S ribosomal subunit
Inhibits protein synthesis by binding
50S ribosomal subunit
Inhibition of protein synthesis by
binding to 50S ribosomal subunit
Bind to 50S ribosomal subunit to
interfere with initiation of protein
synthesis
Inhibit protein synthesis by binding to
two separate sites on the 50S
ribosomal subunit
Inhibit DNA synthesis by binding DNA
gyrases
Inhibits RNA synthesis by binding
DNA-dependent, RNA polymerase
Interfere with folic acid pathway by
binding the enzyme
dihydropteroate synthase
Interferes with folic acid pathway by
binding the enzyme dihydrofolate
reductase
Exact mechanism uncertain; may have
several bacterial enzyme targets
and directly damage DNA
Binding and disruption of cell
membrane
Disruption of cell membrane
Nuclei Acid Synthesis
Sulfonamides
Trimethoprim
Quinolones
Rifampicin
Page |6
PRIMARY PURPOSE
bacilli
Spectrum of Activity
Both gram-positive and gram-negative
bacteria, but spectrum may vary
with the individual antibiotic
Gram-positive and gram-negative
bacteria; not anaerobic bacteria
Wide spectrum of gram-positive and
gram-negative species including
those resistant to tetracycline
Gram-positive and gram-negative
bacteria, and several intracellular
bacterial pathogens (e.g.,
Chlamydia)
Gram-positive and gram-negative
bacteria
Gram-positive cocci including certain
macrolide-resistant strains and
some fastidious gram-negatives
(e.g., H. influenzae and M.
catarrhalis)
Wide variety of gram-positive bacteria,
including those resistant to other
antimicrobial classes
Primarily gram-positive bacteria
Gram-positive and gram-negative
bacteria, but spectrum may vary
with individual antibiotic
Gram-positive and certain gram-
negative (e.g., N.meningitidis
bacteria)
Gram-positive and many gram-
negative bacteria
Gram-positive and many gram-
negative bacteria
Gram-positive and gram-negative
bacteria
Gram-positive bacteria including those
resistant to beta-lactams and
glycopeptides
Gram-negative bacteria, poor activity
against most gram-positive
bacteria
Cell Membrane Function
Polymyxins: colistin
 Page |7

Glycopeptides: Chloramphenicol
vancomycin, teicoplanin Erythromycin
Cycloserine Fusidic acid
Bacitracin

20. C. TRACHOMATIS: Cell culture is the reference method for diagnosis of chlamydial infections and should be
performed when the diagnosis is disputed and in cases of suspected sexual assault or abuse. Cell lines most
commonly used are McCoy or buffalo green monkey cells . (Henry)

21. Direct Fluorescent Antibody Tests. The DFA test allows direct visualization of C. trachomatis elementary bodies
in smears of clinical specimens. (Henry)

22. Enzyme immunoassays: EIAs detect chlamydial LPS with monoclonal or polyclonal antibodies labeled with an
enzyme that converts a colorless substrate into a colored product. (Henry)

23. Nucleic Acid Hybridization Tests. A commercially available acridinium-esterlabeled DNA probe complementary
to C. trachomatis ribosomal RNA allows direct detection of C. trachomatis in urogenital and conjunctival specimens.
(Henry)

24. Nucleic Acid Amplification. Several nucleic acid amplification tests for direct detection of C. trachomatis in
endocervical swab specimens, male urethral swab specimens, and male and female urine samples are commercially
available. (Henry)

25. The gold standard serologic test for rickettsioses is the indirect immunofluorescent antibody (IFA) assay (Kaplan,
1986). The indirect immunoperoxidase antibody test yields similar results. For spotted fever and typhus-group
rickettsial infections in the United States, IFA titers of 1 : 64 or greater are considered to be diagnostic in a compatible
clinicoepidemiologic situation. (Henry)

26. Confirmation with a more specific method, such as microimmunofluorescence, is necessary to detect serum
antibodies to rickettsia. In this procedure, antigenic dots for rickettsial diseases are fixed on a microscope slide, and
the patients serum is added. A fluorescein-conjugated antihuman globulin is added, which binds to the antigen-
antibody complex in a positive reaction. (Delost)

27. Zygomycetes
Coarse, woolly, fluffy, white to gray to brown mycelium with black or brown sporangium. Hyphae grow within 1 - 3 days
and rapidly cover agar surface.
Rhizopus Absidia Mucor
Large, broad, nonseptate hyphae that Similar to Rhizopus; however, No rhizoids.
produce horizontal runners, or stolons, sporangiophores arise between nodes
which attach at rhizoids. from which rhizoids are formed.
Sporangiophores arise in clusters at
rhizoids and terminate sporangia.

28. Morphologically, Geotrichum candidum, exhibits round, oval or rectangular yeast cells that are paired or in chains.
Fragmented hyphae with rectangular arthroconidia with rounded ends when grown on cornmeal agar.

29. Alternaria
Rapidly growing, cottony, gray to black colonies
Septate, dematiaceous conidiophores that may branch with chains of brown conidia, which are muriform and tapered.
 Page |8

30. The greatest hazard for laboratory personnel comes from handling mould cultures of the dimorphic
pathogens, Coccidioides spp. and H. capsulatum. These isolates are classified as risk group 3 pathogens,
requiring biosafety level 3 practices and facilities for propagating and manipulating cultures, as well as for processing
soil or other environmental materials known or likely to contain infectious conidia from these agents. (Henry)

31. List of Viral Syndromes and Common Viral Pathogens

VIRAL SYNDROME VIRAL PATHOGENS
INFANTS AND CHILDREN
Upper respiratory tract Rhinovirus, coronavirus, parainfluenza, adenovirus, respiratory syncytial virus,
infection influenza
Pharyngitis Adenovirus, coxsackie A, herpes simplex, Epstein-Barr virus, rhinovirus,
parainfluenza, influenza
Croup Parainfluenza, respiratory syncytial, metapneumovirus
Bronchitis Parainfluenza, respiratory syncytial, metapneumovirus
 Page |9

VIRAL SYNDROME VIRAL PATHOGENS

Bronchiolitis Respiratory syncytial, parainfluenza, metapneumovirus
Pneumonia Respiratory syncytial, adenovirus, influenza, parainfluenza
Gastroenteritis Rotavirus, adenovirus 40-41, calicivirus, astrovirus
Congenital and neonatal HSV-2, echovirus, and other enteroviruses, CMV, parvovirus B19, VZV, HIV,
disease hepatitis viruses
ADULTS
Upper respiratory tract Rhinovirus, coronavirus, adenovirus, influenze, parainfluenza, Epstein-Barr
infection
Pneumonia Influenza, adenovirus, sin nombre (hantavirus), SARS coronavirus
Pleurodynia Coxsackie B
Gastroenteritis Noroviruses
ALL PATIENTS
Parotitis Mumps, parainfluenza
Myocarditis/pericarditis Coxsackie B and echovirus
Keratitis/conjunctivitis Herpes simplex, varicella-zoster, adenovirus, enterovirus 70
Pleurodynia Coxsackie B
Herpangina Coxsackie A
Febrile illness with rash Echoviruses and coxsackie viruses
Infectious Epstein-Barr virus and cytomegalovirus
mononucleosis
Meningitis Echoviruses and coxsackie viruses, mumps, lymphocytic chroiomeningitis, HSV-2
Encephalitis HSV-1, togaviruses, bunyaviruses, flaviviruses, rabies, enteroviruses, measles, HIV,
JC virus

Hepatitis Hepatitis A, B, C. D (delta agent), E, and non-A, B, C, D, E

Hemorrhagic cystitis
Cutaneous infection with or
without rash
Hemorrhagic fever
Generalized, no specific
target organ
Adenovirus, BK virus
HIS-1 and 2, varicella-zoster, enteroviruses, measles, rubella, parvovirus B-19,
human herpesvirus 6 and 7, HPV, poxviruses including smallpox, monkeypox,
molluscum contagiosum, and orf
Ebola, Marburg, Lassa, yellow fever, dengue and other viruses
HIV-1, HIV-2, HTLV-1

32. Rubella (German measles) is highly communicable and produces mild fever and a transient rash in children and
adults. All infections are viremic, and transplacental spread during the first trimester produces devastating
teratogenic cardiac, ocular, and brain malformations. Routine prenatal screening for maternal rubella IgG as proof of
immunity is standard practice. When acute rubella is suspected in a pregnant woman, the most straightforward
diagnostic method is assay of maternal serum for rubella IgM by EIA or IFA. Culture of rubella is technically complex
and is not routinely available. The congenitally infected newborn is IgM positive and excretes rubella in urine for
months to years. (Henry)

33. ROTAVIRUS
EIA is used to detect rotavirus antibody and for serotyping
EIA and latex agglutination can be used for detection of group A rotavirus antigen in fecal samples
RT-PCR can detect rotavirus RNA

34. Acanthamoeba keratitis is an increasingly recognized painful infection of the cornea that is most likely to occur in
persons who use daily-wear or extended-wear soft contact lenses or who have experienced trauma to the cornea.
Incomplete or infrequent disinfection and use of homemade saline and multipurpose solutions are known risk factors
for acquiring the infection. (Henry)

35. COMPARISON OF MORPHOLOGICAL FEATURES OF MALARIA PARASITES (Belizario, de Leon)

Parameter P. vivax P. malariae P. falciparum P. ovale
(benign tertian) (quartan) (malignant tertian)
Infected red Larger than normal, Normal or slightly Normal, multiple Somewhat larger than
blood cells pale, often bizarre; smaller; sometimes infection of RBC very normal, often with
Schuffner dots are often darker in early stages; common fringed or irregular edge,
present; multiple multiple infections of and oval in shape;
infection of RBC not RBC are rare Schuffners dots appear
uncommon even with younger

Parameter P. vivax
(benign tertian)
Small Signet-ring form with
trophozoites heavy red dot and blue
(early rings) cytoplasmic ring
Growing Like small trophozoite,
trophozoite as above, with
increased cytoplasm
and ameboid activity;
small yellowish-brown
pigment granules in
cytoplasm; increasing
with age of parasite
Large Large mass of
trophozoite chromatin; loose,
irregular, or close
compact cytoplasm with
increasing amount of
fine brown pigment;
parasite fills cell in 30 to
40 hours
Schizont Chromatin divided;
(pre- cytoplasm shows
segmenting) varying degrees of
separation into strands
and particles; pigment
collects in parts of the
parasite
Schizont 12-24 merozoites;
(mature) pigment in 1-2 clumps;
parasite almost fills
enlarged cells
Gametocyte Microgametocyte: light
red to pink chromatin,
diffuse, central; gives tint
to light blue cytoplasm;
yellowish brown pigment
throughout cytoplasm;
usually round and about
the size of normal RBC
Macrogametocyte:
small, compact, dark red
eccentric chromatin;
cytoplasm dark blue, no
vacuoles; abundant dark
brown pigment scattered
throughout the cytoplasm
Stages in All stages are present
peripheral
blood
Length of 48 hours
asexual
cycle
P. malariae
(quartan)
Same as P. vivax but
with blue cytoplasmic
circle, smaller, thicker
and heavier
Chromatin rounded or
elongated; cytoplasm
compact or in narrow
band across cell; dark
brown granules may
have peripheral
arrangement
Chromatin often
elongate, indefinite in
outline; cytoplasm
dense; compact, in
rounded oblong or band
forms; pigment granules
larger, darker than P.
vivax; parasite fills cells
frequently
Same as P. vivax except
parasite is smaller,
shows less chromatin
division, more delayed
clumping of pigment
6-12 (average 8-10)
merozoites in rosette
form; parasite almost
fills cell
Same as P. vivax except
smaller; fills or almost
fills cells
All stages are present
72 hours
P a g e | 10
P. falciparum P. ovale
(malignant tertian)
stages; stains more
readily and deeply than
in P. vivax
Same as P. vivax but Small, darker in color,
with small threadlike and generally more solid
blue cytoplasmic circle than those of P.
with 1 or 2 small red falciparum; Schuffners
chromatin dots; double dotes regularly present
chromatin common; in almost 100% of
marginal forms common infected cells
Remains in ring form but Resembles closely
grows resembling small same stage of P.
trophozoite of P. vivax in malariae but is
size; usually the oldest considerably larger;
asexual stage seen in pigment is lighter and
peripheral blood less conspicuous
Seldom present Seldom present
Not present About 25% of infected
cells are definitely oval
shaped; usual picture is
that of a round parasite
in the center of an oval
cell, many cells with
indefinite fringed outline;
pigment lighter and less
coarse than in P.
malariae
Rarely present; 8-24 Usually 8 merozites
merozoites smaller than arranged a central block
other species of pigment
Present in peripheral Distinguished from P.
bloodstream; similar to malariae by size of
P. vivax; crescent or infected cells and by
sausage-shaped Schuffners dots; less
easy to differentiate
from P. vivax
Ring forms and All stages are present
gametocytes; other
stages rare
48 hours or less 48 hours
 P a g e | 11

36. DNA stores human genetic information and dictates the amino acid sequence of peptides and proteins. The purine
bases (adenine and guanine) and the pyrimidine bases (cytosine and thymine). Adenine pairs with thymine with two
hydrogen bonds and cytosine pairs with guanine with three hydrogen bonds. The strands are complementary due to
the fixed manner by which the base pairs bond.

37. PCR is a simple in vitro chemical reaction that permits the synthesis of essentially limitless quantities of a targeted
nucleic acid sequence. This is accomplished through the action of a deoxyribonucleic acid (DNA) polymerase that,
under the right conditions, can copy a strand of DNA. At its simplest, a PCR consists of target DNA, a molar excess of
two oligonucleotide primers, a heat-stable DNA polymerase, an equimolar mixture of deoxyribonucleotide
triphosphates (dATP, dCTP, dGTP, and dTTP), MgCl2, KCl, and a Tris-HCl buffer. The two primers flank the sequence
to be amplified, are typically 1830 bases long, and are complementary to opposite strands of the target.

To initiate a PCR, the reaction mixture is heated to separate the two strands of target DNA (denaturation) and then
cooled to permit the primers to anneal to the target DNA in a sequence-specific manner (annealing). The DNA
polymerase then initiates extension of each primer at its 3 end (extension). The primer extension products are
dissociated from the target DNA by heating. Each extension product, as well as the original target, can serve as a
template for subsequent rounds of primer annealing and extension.

A PCR cycle consists of three steps: denaturation, annealing, and extension. At the end of each cycle, the
PCR products are theoretically doubled.

38. PCR is a licensed technology, and other methods of nucleic acid amplification have since been developed. In
branched DNA (bDNA) signal amplification, the target DNA is denatured and added to a well containing immobilized
probes. One end of each probe hybridizes with the target DNA, capturing it, and the other contains multiple branches
that hybridizes with the target DNA, capturing it, and the other contains multiple branches that hybridize with alkaline
phosphatase-labeled probes. After washing to remove the unbound labelled probes, dioxetane is added, and
chemiluminescence is measured. A thermocycler is not used and the target DNA is not amplified. Other nucleic acid
amplification methods include nucleic acid sequence-based amplification (NASBA), transcription-mediated
amplification (TMA), hybrid capture, and rolling circe; amplification (RCA). HARR: the target DNA is bound by
multiple probes, and those are amplified instead of the target DNA.

39. No genomic or plasmid DNA, RNA, or patient specimens should be introduced into the dead air box where reagent
preparation takes place. This dead air box should be furnished with an ultraviolet (UV) light connected to a timer. The
surface areas of the dead air boxes should be treated with UV light, then wiped with freshly made 10% sodium
hypochlorite solution and finally with 75% ethanol solution before and after working in this area. (Henry)

CLINICAL CHEMISTRY

40. WAVELENGTH SELECTORS: A critical component of all spectrometers is the device used to select the appropriate
wavelength. Several types of wavelength selectors are available, including filters, prisms, grating monochromators,
and, recently, holographic gratings. (Henry)

41. Monochromator disperses the light into wavelengths. Glass filters and interference filters are used in photometers.
Diffraction gratings and prisms are used in spectrophotometers. (Ciulla)

42. Fluoresecence is a process where atoms absorb energy at a particular wavelength (excitation), electrons are raised
to higher-energy orbitals, and the electrons release energy as they return to ground state by emitting light energy if a
longer wavelength and lower energy than the exciting wavelength. (Ciulla)

43. COLOR REACTION FOR QUANTITATION OF BILIRUBIN

Bilirubin + diazotized sulfanilic acid azobilirubin
Color is proportional to the concentration of bilirubin

ASSAY EVELYN-MALLOY JENDRASSIK-GROF

pH Acid Alkaline
Dissociating agent Methanol Caffeine-sodium benzoate
Diazo product Red or reddish-purple color Blue (maximum absorbance around 600
(absorption maximum in the region nm)
of 560 nm)

44. Malloy and Evelyn developed the first clinically useful methodology for the quantitation of bilirubin in serum samples
using the classic diazo reaction with a 50% methanol solution as an accelerator. The Jendrassik and Grof described a
method using the diazo reaction with caffeine-benzoate-acetate as an accelerator. Today, all commonly used methods
for measuring bilirubin and its fractions are modifications of the technique described by Malloy and Evelyn. Total
bilirubin and conjugated bilirubin are measured and unconjugated bilirubin is determined by subtracting conjugated
bilirubin from total bilirubin. (Bishop)

45. BILIRUBIN FRACTIONS

Conjugated bilirubin Contains one or two attached glucuronic acid molecules
Reacts directly with the color reagent
 P a g e | 12

Also referred to as DIRECT BILIRUBIN

Unconjugated Noncovalently attached to albumin
bilirubin Does not react with the color reagent until the bilirubin is first dissociated from the protein
INDIRECT BILIRUBIN
Delta bilirubin Bilrubin fraction that is covalently attached to protein
Reacts directly with the color reagent and contributes to the direct, or conjugated, bilirubin
value

46. Glycosylated hemoglobin assays vary in reliability in the presence of a variety of factors. Interference by carbamylated
hemoglobin can occur with uremia, hypertriglyceridemia, and hyperbilirubinemia, and salicylates can cause
interference by acetylated species. Hemoglobinopathies (HbSS, HbSC, HbCC) associated with high red blood cell
turnover and the need for transfusions will adversely affect accuracy, as will chronic alcohol or opiate use, iron
deficiency, and lead poisoning. Vitamins C and E can falsely lower levels by inhibiting glycosylation, but vitamin C can
also increase levels for some assays. Sample storage effects may occur. Any condition associated with
shortened red blood cell survival or lower mean red blood cell age, such as hemolysis, recovery from acute
blood loss, transfusions, or splenectomy, will lower the HbA1c level as the result of reduced exposure to
plasma glucose. Hyperglycemia has been associated with a decrease in erythrocyte survival, suggesting that HbA1c
levels in poorly controlled patients may underestimate their mean plasma glucose concentration. (Henry)

47. Several analytic approaches have been used to assay for urea. Enzymatic methods are used most frequently in
clinical laboratories. The enzyme urease (urea amidohydrolase) hydrolyzes urea in the sample and the ammonium ion
(NH4+) produced in the reaction is quantified.The most common method couples the urease reaction with glutamate
dehydrogenase (GLDH) and the rate of disappearance of nicotinamide adenine dinucleotide (reduced, NADH) at 340
nm is measured. (Bishop)

48. Total Iron Content (Serum Iron). Measurement of serum iron concentration refers specifically to the Fe +3 bound to
transferrin and not to the iron circulating as free hemoglobin in serum. Spectrophotometric determinations have been
adapted to automated analysis. These procedures generally have the following steps: Fe +3 is released from binding
proteins by acidification, reduced to Fe_2 by ascorbate or a similar reducing agent, and complexed with a color
reagent such as ferrozine, ferene, or bathophenanthroline. (Bishop)

49. Serum (Total) Iron-Binding Capacity. The reference interval for adults is 250400 g/dL (4572 mol/L). In iron
deficiency anemia, the serum TIBC is increased. It is normal or decreased in the anemia of chronic disease. (Henry)

50. CHARACTERISTICS OF SELECTED PLASMA PROTEINS (Bishop 6th Ed.)

Prealbumin (Transthyretin) INDICATOR OF MALNUTRITION; binds thyroid hormones and retinol-binding protein
Albumin Binds bilirubin, steroids, fatty acids; major contributor to oncotic pressure
1-Globulins
1-antitrypsin Acute-phase reactant; protease inhibitor
1-fetoprotein Principal fetal protein
1-acid glycoprotein Acute-phase reactant
(orosomucoid)
1-lipoprotein (HDL) Transports lipid
1-antichymotrypsin Inhibits serine proteinases (ie, chymotrypsin)
Inter--trypsin inhibitor Inhibits proteinases (ie, trypsin)
Gc-globulin Binds vitamin D and actin
2-Globulins
Haptoglobins Acute-phase reactant; binds hemoglobin
Ceruloplasmin Peroxidase activity; contains copper
2-Macroglobulin Inhibits thrombin, trypsin, pepsin
-Globulins
Pre--Lipoproteins (VLDL) Transports lipids (primarily triglyceride)
Transferrin (Siderophilin) Transports iron
Hemopexin Binds heme
-Lipoproteins (LDL) Transports lipids (primarily cholesterol)
2-Micorglobulin (B2M) Component of human leukocyte antigen (HLA) molecules class I
Complement Immune response
Fibrinogen Precursor of fibrin clot
C-reactive protein (CRP) Acute-phase reactant; motivates phagocytosis in inflammatory disease (Bishop)
-Globulins
Immunoglobulin G Antibodies
Immunoglobulin A Antibodies (in secretions)
Immunoglobulin M Antibodies (early response)
Immunoglobulin D Antibodies
Immunoglobulin E Antibodies (allergy)
 P a g e | 13

51. CRYOGLOBULINEMIA: Cryoglobulins are Igs that reversibly precipitate at cold temperature. Three categories
of cryoglobulins exist. Type I cryoglobulin consists of a single monoclonal protein and is usually seen in multiple
myeloma, Waldenstrms macroglobulinemia, or other lymphoproliferative disorders. Usually, the immunoglobulin is
IgG or IgM. Type II cryoglobulin consists of a mixture of polyclonal immunoglobulins with a monoclonal protein, usually
IgM, directed toward polyclonal IgG. Type II cryoglobulinemia is associated with chronic hepatitis C infection,
autoimmune disorders, and IgM-producing malignancies. Finally, Type III cryoglobulin consists of polyclonal
immunoglobulins, usually IgM, that have activity toward polyclonal IgG. They are associated with autoimmune
conditions, as well as infections. Cryoglobulins produce symptoms by precipitating in areas of low temperature, such
as the skin or extremities, resulting in vascular occlusion. This produces a wide variety of symptoms, including
acrocyanosis, Raynauds phenomenon, skin ulcers, skin purpura, and glomerulonephritis (Henry)

52. The role of magnesium in the body is widespread. It is an essential cofactor of more than 300 enzymes, including
those important in glycolysis, transcellular ion transport, neuromuscular transmission, synthesis of carbohydrates,
proteins, lipids, and nucleic acids, and release of and response to certain hormones. (Bishop)

53. The main biochemical role of zinc is its influence on the activity of more than 300 enzymes (from the classes of
oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases). (Bishop)

54.MAJOR ENZYMES OF CLINICAL SIGNIFICANCE

ENZYME CLINICAL SIGNIFICANCE
 P a g e | 14

1. Acid phosphatase (ACP) Prostatic carcinoma

2. Alanine aminotransferase (ALT) Hepatic disorder
3. Aldolase (ALD) Skeletal muscle disorder
4. Alkaline phosphatase (ALP) Hepatic disorder
Bone disorder
5. Amylase (AMS) Acute pancreatitis
6. Angiotensin-converting enzyme (ACE) Blood pressure regulation
7. Aspartate aminotransferase (AST) Myocardial infarction
Hepatic disorder
Skeletal muscle disorder
8. Chymotrypsin (CHY) Chronic pancreatitis insufficiency
9. Creatine kinase (CK) Myocardial infarction
Skeletal muscle disorder
10. Elastase-1 (E1) Chronic pancreatitis insufficiency
11. Glucose-6-phosphate dehydrogenase (G-6-PD) Drug-induced hemolytic anemia
12. Glutamate dehydrogenase (GLD) Hepatic disorder
13. -Glutamyltransferase (GGT) Hepatic disorder
14. Glutathione-S-transferase (GST) Hepatic disorder
15. Glycogen phosphorylase (GP) Acute myocardial infarction
16. Lactate dehydrogenase (LDH) Myocardial infarction
Hepatic disorder
Hemolysis
Carcinoma
17. Lipase (LPS) Acute pancreatitis
18. 5-Nucleotidase Hepatic disorder
19. Pseudocholinesterase (PChE) Organophosphate poisoning
Genetic variants
Hepatic disorder
Suxamethonium sensitivity
20. Pyruvate kinase (PK) Hemolytic anemia
21. Trypsin (TRY) Acute pancreatitis

55. CHARACTERISTICS OF ISOENZYMES OF ALKALINE PHOSPHATASE

SOURCE OF ENZYME INHIBITION BY ORDER
L-phenylalanine (%) Heat or Urea (%) ANODAL MIGRATION
Liver 10 60 1
Bone 10 90 2
Intestine 75 60 4
Placenta 80 0 3
Regan (carcinoma) 80 0 3

Measurement of Total ALP Activity

REACTION NAME SUBSTRATE USED COMMENTS
Shinowara-Jones-Reinhart Beta-glycerophosphate Long incubation time; high blank
values
King-Armstrong Phenylphosphate Endpoint, requires protein removal
Bessey-Lowry-Brock p-Nitrophenylphosphate Endpoint or kinetic, rapid
Bowers-McComb p-Nitrophenylphosphate Uses phosphate-accepting buffer;
reference method

56. Measurement of Total ACP Activity

REACTION NAME SUBSTRATE USED COMMENTS
Bodansky Beta-glycerophosphate Lengthy assay, nonspecific
Gutman, King-Armstrong Phenylphosphate Nonspecific
Hudson p-Nitrophenylphosphate Rapid, nonspecific
Babson and Reed Alpha-naphthylphosphate Complicated, less sensitive
Roy Thymolphthalein monophosphate More specific for prostatic form
Rietz, Guilbault 4-Methylumbelliferonephosphate Fluorescent, some improved sensitivity

57. ENZYMES IN CARDIAC DISORDERS

AMI occurs when there is abrupt reduction in blood flow to a region of myocardial tissue, usually caused by
atherosclerosis of the coronary arteries
Enzyme Onset of Elevation (h) Peak Activity (h) Duration of Elevation (days)
CK 4-8 12-24 3-4
AST 8-12 24 5
LD 12-24 72 10
 P a g e | 15

58. Methods used for measurement of CK isoenzymes include electrophoresis; ion-exchange chromatography and
several immunoassays, including radioimmunoassay (RIA) and immunoinhibition methods. Although mass methods
are more sensitive and preferred for quantitation of CK-MB, electrophoresis has been the reference method. (Bishop)

59. Immunoassays detect CK-MB reliably with minimal cross-reactivity. Immunoassays measure the concentration of
enzyme protein rather than enzymatic activity and can, therefore, detect enzymatically inactive CK-MB. This leads to
the possibility of permitting detection of infarction earlier than other methods. A double-antibody immunoinhibition
assay is also available. This technique allows differentiation of MB activity due to adenylate kinase and the atypical
isoenzymes, resulting in a more specific analytic procedure for CK-MB. Point-of-care assay systems for CM-MB are
available but not as widely used as those for troponins. (Bishop)

60. In the immunoinhibition technique for CKMB determination, antibodies are directed against the M and B units of the
enzymes. Anti-M inhibits all M activity but not B activity. CK activity is measured before and after inhibition. The
activity remaining after inhibition is a result of the B subunit for BB and MB activity. (BOR)

61. Cardiac troponin T or cardiac troponin I is used as an acute myocardial infarction indicator because of specificity and
early rise in serum concentration following AMI. In cases of AMI, cTnT increases 3 to 4 hours following infarction,
peaks in 10 to 24 hours, and returns to normal in 10 to 14 days. cTnI increases in 3 to 6 hours following infarctions,
peaks in 14 to 20 hours, and returns to normal in 5 to 10 days. (Ciulla)

62. Laboratory diagnosis of acute pancreatitis includes elevation of serum and urine amylase and serum lipase. Serum
AMS levels generally become elevated within 2 hours to 12 hours of an attack, reach peak levels at 24 hours, and
return to normal within 3 to 5 days. Serum LPS peak levels occur at 12 to 24 hours, and levels remain elevated longer
than AMS.
63. Routine measurement of electrolytes usually involves only Na+, K+, Cl-, and HCO3- (as total CO2). These values may
be used to approximate the anion gap (AG), which is the difference between unmeasured anions and unmeasured
cations. (Bishop)

There are two commonly used methods for calculating the anion gap.
The first equation is
AG = Na+ - (Cl- + HCO3-)
The reference range for the AG using this calculation is 716 mmol/L.

The second calculation method is

AG = (Na++ K+) - (Cl- + HCO3-)
It has a reference range of 1020 mmol/L.

An elevated anion gap may be caused by uremia/renal failure, which leads to PO 4- and SO42- retention; ketoacidosis,
as seen in cases of starvation or diabetes; methanol, ethanol, ethylene glycol poisoning, or salicylate; lactic acidosis;
hypernatremia; and instrument error. Low anion gap values are rare but may be seen with hypoalbuminemia
(decrease in unmeasured anions) or severe hypercalcemia (increase in unmeasured cations). (Bishop)

64. All sodium ions must be neutralized by counter-ions, most of which, in blood, are constituted by chloride and
bicarbonate ions, and, to a lesser degree, by phosphate, sulfate, and protein carboxylate groups. Normal serum
sodium is about 140 mEq/L, chloride is usually around 100 mEq/L, and bicarbonate around 2 mEq/L. The anion gap
is then defined as Na+ (Cl + HCO3), which for normal individuals is around 16. This 16 mEq/L really accounts
for the other counter-ions that neutralize sodium but are not measured in serum.

65. DETERMINATION OF LACTATE: Special care should be practiced when collecting and handling specimens for
lactate analysis. Ideally, a tourniquet should be not be used because venous stasis will increase lactate levels. If a
tourniquet is used, blood should be collected immediately and the patient should not exercise the hand before or
during collection.14 After sample collection, glucose is converted to lactose by way of anaerobic glycolysis and should
be prevented. Heparinized blood may be used but must be delivered on ice and the plasma must be quickly
separated. Iodoacetate or fluoride, which inhibit glycolysis without affecting coagulation, are usually satisfactory
additives, but the specific method directions must be consulted. (Bishop)

66. METABOLIC ACIDOSIS

LABORATORY FINDINGS IN METABOLIC ACIDOSIS LABORATORY FINDINGS IN COMPENSATION
ctCO2 decreased ctCO2 decreased
PCO2 normal PCO2 decreased
pH decreased pH normal

67. METABOLIC ALKALOSIS

LABORATORY FINDINGS IN METABOLIC ALKALOSIS LABORATORY FINDINGS IN COMPENSATION
ctCO2 increased ctCO2 increased
PCO2 normal PCO2 increased
pH increased pH normal

68. RESPIRATORY ACIDOSIS

 P a g e | 16

LABORATORY FINDINGS IN RESPIRATORY ACIDOSIS LABORATORY FINDINGS IN COMPENSATION

ctCO2 normal ctCO2 increased
PCO2 increased PCO2 increased
pH decreased pH normal

69. RESPIRATORY ALKALOSIS

LABORATORY FINDINGS IN RESPIRATORY LABORATORY FINDINGS IN COMPENSATION
ALKALOSIS
ctCO2 normal ctCO2 decreased
PCO2 decreased PCO2 decreased
pH increased pH normal

70. THERAPEUTIC DRUGS

CARDIOTROPICS Digitalis glycosides: digoxin and digitoxin
Most commonly used to treat congestive heart failure and Procainamide (Pronestyl)
cardiac arrythmias Quinidine
Lidocaine (Xylocaine)
Propranolol
Disopyramide
ANTICONVULSANTS Phenobarbital
Used in the treatment of seizure disorders, in particular Phenytoin (Dilantin)
grand mal, petit mal, and psychomotor seizures and other Primidone (Mysoline)
generalized seizure disorders such as tic douloreux Ethosuximide (Zarontin)
(trigeminal neuralgia) Carbamazepine (Tegretol)
Valproic acid (Depakene)
ANTIASTHMATICS Theophylline
Most commonly prescribed anti-asthmatic drug
Bronchodilator for the treatment of moderate to severe
asthma, both for the prevention of attacks and for treatment
of symptomatic exacerbations
ANTI-INFLAMMATORY DRUGS Acetaminophen (Tylenol)
Acetylsalicylic acid
IMMUNOSUPPRESSIVES Cyclosporine
Prednisone
Cyclophosphamide (Cytoxan)
CHEMOTHERAPEUTIC AGENTS Methothrexate
An anti-neoplastic agent
Important immunosuppressive agent used in the treatment
of psoriasis, rheumatoid arthritis, and some collagen
vascular diseases
DRUGS FOR TREATMENT OF MANIC-DEPRESSION Lithium anti-manic agent and are used for the
Used in the treatment of psychiatric affective disorders prophylaxis and treatment of bipolar disorder (manic-
depressive psychosis)
Tricyclic Antidepressants: Amitriptyline, Imipramine,
Nortriptyline, Desipramine, Doxepin

71. PROCAINAMIDE: Like quinidine, procainamide is used to treat cardiac arrhythmia. Oral administration is the most
common. Gastrointestinal absorption is rapid and complete. Peak plasma concentrations occur at about 1 hour.
Absorbed procainamide is about 20% bound to plasma proteins. It is eliminated by a combination of renal filtration
and hepatic metabolism. N-Acetyl procainamide (NAPA) is a hepatic metabolite of the parent drug, with
antiarrhythmic activity similar to procainamide. The total antiarrhythmic potential of this drug must take into
consideration the parent drug and this metabolite. Alteration in either renal or hepatic function may lead to increased
serum concentration of the parent drug and its metabolites. Increased concentration results in myocardial
depression and arrhythmia. Both procainamide and its active metabolite can be measured by immunoassay.
(Bishop)

72. Cocaines short half-life is a result of rapid hepatic hydrolysis to inactive metabolites. This is the major route of
elimination. Only a small portion of the parent drug can be found in urine after an administered dose. The primary
product of hepatic metabolism is benzoylecgonine, which is primarily eliminated in urine. The half-life of
benzoylecgonine is 47 hours. The presence of this metabolite in urine is a sensitive and specific indicator of
cocaine use. It can be detected in urine for up to 3 days after a single use. In chronic heavy abusers, it can be
detected in urine for up to 20 days after the last dose. The primary screening procedure for identification of cocaine
use is detection of benzoylecgonine in urine by immunoassay.
Confirmation testing is done by GC/MS. (Bishop)
 P a g e | 17

73. Serum HER2/neu is used as marker of prognosis and monitoring therapy for breast cancer. HER2/neu is
cancerspecific but not tissue-specific and is found to be increased in breast cancers, as well as lung and other
epithelial cell tumors. (Henry)

74.RECEPTOR TUMOR MARKERS

TUMOR MARKER TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
Estrogen receptor Breast cancer IHC Biopsy Hormonal therapy
indicator
Progesterone receptor Breast cancer IHC Biopsy Hormonal therapy
indicator
Her-2/neu Breast, ovarian, IHC, FISH, ELISA Biopsy Prognostic and
gastrointestinal tumors hormonal therapy
indicator
Epidermal growth Head, neck, ovarian, IHC Biopsy Prognostic indicator
factor receptor cervical cancers
ELISA, enzyme linked immunosorbent assay; FISH, fluorescence in situ hybridization;
IHC, immunohistochemistry.

HEMATOLOGY

75. Red blood cells that resist lysis, such as sickle cells, can produce falsely elevated WBC counts and falsely increase
the number of lymphocytes. (Steininger)

76. The electrophoretic mobility of hemoglobin D is the same as hemoglobin S, although red blood cells containing
hemoglobin D show no sickling at a reduced oxygen concentration. (Brown)

77. Morphologic Findings in Red Cells

Finding Definition Associated Conditions
Basophilic Small blue dots in red cells, due to clusters of ribosomes. Hemolytic anemias, lead poisoning,
stippling thalassemia
Pappenheimer Larger, more irregular, and grayer than basophilic stippling, Asplenia, sideroblastic anemia
bodies due to iron-containing mitochondria.
Heinz bodies Heinz bodies: gray-black inclusions, seen only with Oxidative injury, G6PD deficiency or
Bite cells supravital stains (crystal violet). Bite cells: sharp bite-like unstable hemoglobin
defects in red cells where a Heinz body has been removed
in the spleen. Both are due to denatured hemoglobin.
Howell-Jolly Howell-Jolly body: dot-like dark-purple inclusion. Cabot Asplenia
bodies ring: ring shaped dark purple inclusion. Both represent a
Cabot Rings residual nuclear fragment.
Target cells Red cells with dark circle within the central area of pallor, Thalassemia, hemoglobin C, liver
reflecting a redundant membrane. disease
Schistocytes Fragmented red blood cells, taking shapes such as helmet- Microangiopathic haemolytic anemias
shaped cells, due to mechanical red cell fragmentation. (MHA), DIC, TTP, HUS, mechanical
heart valves
Dacrocytes Teardrop or pear-shaped erythrocytes. Can be seen in relatively benign
(teardrop cells) conditions (thalessemia, megaloblastic
anemia), often seen in myelophthisis
Echinocytes Red blood cells that have circumferential undulations or Uremia, gastric cancer, pyruvate kinase
(burr cells) spiny projections with pointed tips. deficiency
Acanthocytes Red blood cells that have circumferential blunt and spiny Liver disease, abetalipoproteinemia,
(spur cells) projections with bulbous tips. McLeod phenotype
Spherocytes Red cells without central pallor due to decreased red cell Immune haemolytic anmeia, hereditary
membrane. spherocytosis
Elliptocytes Red cells twice as long as they are wide. Iron deficiency, hereditary elliptocytosis
Stomatocytes Red cells whose area of central pallor is elongated in a Alcohol, Dilantin, Rh null phenotype,
mouth-like shape. hereditary stomatocytosis

78. Spherocytic red blood cells are almost spherical in shape. They are not biconcave like a normal red blood cells
and do not have the central area of pallor which a normal red cell shows. These cells are associated with hemolytic
anemia, ABO haemolytic disease of the newborn, and hereditary spherocytosis. (Brown)

79. Stomatocytes show a slit-like area of central pallor. These red blood cells have lost the indentation on one side and
may be found un liver disease, alcoholism, electrolyte imbalance, and hereditary stomatocytosis. (Brown)

80. Teardrop red blood cells or dacrocytes are found most notably in myelofibrosis, pernicious anemia, myeloid
metaplasia, thalassemia and some hemolytic anemias.

81. LABORATORY FEATURES IN MICROCYTIC HYPOCHROMIC ANEMIAS

Marrow
Serum Serum % % Iron Serum ZPP Hb Hb
 P a g e | 18

iron TIBC Saturation Sideroblasts stores ferritin A2 F

Iron deficiency N N

-Thalassemia trait N () N N N N N N N

Anemia of chronic N N N N N
disease
Sideroblastic anemia N N
()
TIBC = total iron-binding capacity; ZPP = zinc protoporphyrins; = decreased; N = normal; = increased.
83. Anemia of chronic disease (ACD) designates an anemia syndrome typically found in patients with chronic infections
or inflammatory or neoplastic disorders; it is characterized by reduced reticulocyte response accompanied by low
serum iron, despite adequate iron stores.The serum iron concentration is characteristically decreased, the TIBC is
decreased or normal (in contrast to iron deficiency anemia, in which the TIBC is elevated), and the percent
saturation is decreased. Erythrocyte protoporphyrin and serum ferritin are elevated. (Henry)

84. An alpha thalassemia of varying severity results from reduced of absent alpha chain synthesis affecting the two
pairs of alpha globin genes (one pair in each chromosome. As the capacity to produce chains decreases, this
results in an accumulation of excess chains in fetal life to form hemoglobin Barts (4) and an excess of chains to
form hemoglobin H (4) later on. (Brown)

85. The presence of Heinz bodies in freshly drawn blood indicates that (1) an oxidizing drug or chemical (e.g.,
phenylhydrazine, chlorate, naphthalene, dapsone) has been ingested in sufficient amount to overwhelm the normal
protective mechanisms of the red cell and denature Hb; (2) a drug such as primaquine has been ingested by an
individual with G6PD deficiency (or another defect resulting in a deficiency of reduced glutathione), so that Hb is not
protected from oxidative denaturation; or (3) the subject has an unstable Hb. (Henry)

86. Acquired or pseudo-Pelger-Huet anomaly is most often seen in chronic myelogenous leukemia and myeloid
metaplasia but may also be seen in patients receiving chemotherapy. Most of these pseudo-Pelger-Huet cells have
a round, centrally located nucleus. The acquired form can also be differentiated from the inherited form by the
presence of more than 10% of normal three lobed neutrophils. (Brown)

87. Cytochemical Stains in Myeloblasts

Type PAS SBB MPO CAE NSE Auer rods
M0 - - - - - Rare
M1 - + + + - 50%
M2 - + + + - 50%
M3 - + + + - 95%
M4 - + + + + 64%
M5 - - - - + 0
M6 + + + - - 50%
M7 + - - - +/- Rare

88. Leukocyte alkaline phosphatase: Segmented neutrophils, neutrophil bands, neutrophil metamyelocytes, as well
as myelocytes may show ALP activity. Only segmented neutrphils are counted. Eosinophils do not stain and must
be recognized by their nuclear structure and by the presence of refractile droplets (cytoplasmic granules) so they
are not included in the count and mistakenly scored as 0. (Steininger)

89. In essential thrombocythemia, the most striking feature is the marked increase in platelets (450 109/L; usually
>1000 109/L), often with abnormal and giant forms, and usually accompanied by fragments of megakaryocytes.
Platelet function defects in thrombocythemia are frequently demonstrable. (Henry)

CLINICAL MICROSCOPY

90. CHANGES IN UNPRESERVED URINE

COLOR Modified or darkened BILIRUBIN Decreased

CLARITY Decreased UROBILINOGEN Decreased
ODOR Increased NITRITE Increased
pH Increased RBCs and WBCs Decreased
GLUCOSE Decreased BACTERIA Increased
KETONES Decreased
 P a g e | 19

91. The pH of freshly excreted urine does not reach 9 in normal or abnormal conditions. A pH of 9 is associated with an
improperly preserved specimen and indicates that a fresh specimen should be obtained to ensure the validity of the
analysis. (Strasinger)

92. Urine specimens containing porphyrins also may appear red resulting from the oxidation of porphobilinogen to
porphyrins. They are often referred to as having the color of port wine. (Strasinger)

93. COMPARISON OF GLUCOSE OXIDASE AND CLINITEST: There are several explanations for the finding of
conflicting results between the two glucose tests. As stated, the Clinitest is not as sensitive as the glucose oxidase
test, so the finding of a 1+ reagent strip reading and a negative Clinitest should not be surprising. A strongly positive
reagent strip and a negative Clinitest, however, should cause concern about possible contamination by strong
oxidizing agents. The most significant discrepancy is the negative reagent strip with a positive Clinitest. Although
interfering substances affecting either test may cause this problem, the most frequent cause is the presence of
other reducing sugars in the urine. (Strasinger)

94. Glycosuria occurs in the absence of hyperglycemia when the reabsorption of glucose by the renal tubules is
compromised. This is frequently referred to as renal glycosuria and is seen in end-stage renal disease, cystinosis,
and Fanconi syndrome. (Strasinger)

95. When RBCs are present, the urine is red and cloudy; however, if hemoglobin or myoglobin is present, the
specimen is red and clear. (Strasinger)

96. Hemoglobinuria may result from the lysis of red blood cells produced in the urinary tract, particularly in dilute, alkaline
urine. It also may result from intravascular hemolysis and the subsequent filtering of hemoglobin through the
glomerulus. Lysis of red blood cells in the urine usually shows a mixture of hemoglobinuria and hematuria, whereas
no red blood cells are seen in cases of intravascular hemolysis. Under normal conditions, the formation of large
hemoglobin-haptoglobin complexes in the circulation prevents the glomerular filtration of hemoglobin. When the
amount of free hemoglobin present exceeds the haptoglobin contentas occurs in hemolytic anemias, transfusion
reactions, severe burns, brown recluse spider bites, infections, and strenuous exercisehemoglobin is available for
glomerular filtration. Reabsorption of filtered hemoglobin also results in the appearance of large yellow-brown
granules of denatured ferritin called hemosiderin in the renal tubular epithelial cells and in the urine sediment.
(Strasinger)

97. MYOGLOBINURIA: The presence of myoglobin rather than hemoglobin should be suspected in patients with
conditions associated with muscle destruction (rhabdomyolysis). Examples of these conditions include trauma,
crush syndromes, prolonged coma, convulsions, muscle-wasting diseases, alcoholism, heroin abuse, and extensive
exertion. The development of rhabdomylosis has been found to be a side effect in certain patients taking the
cholesterol-lowering statin medications. (Strasinger)

98. URINE BILIRUBIN: Questionable results can be repeated using the Ictotest. The Ictotest is less subject to
interference and is sensitive to 0.05 to 0.10 mg/dL of bilirubin, whereas the reagent strips have a lower sensitivity
level of 0.40 mg/dL. Because of its higher sensitivity, an Ictotest may be requested to detect early stages of liver
disease. (Strasinger)

99. Nephrotic syndrome is marked by massive proteinuria (greater than 3.5 g/d), low levels of serum albumin, high
levels of serum lipids, and pronounced edema. Urinalysis observations include marked proteinuria; urinary fat
droplets; oval fat bodies; renal tubular epithelial (RTE) cells; epithelial, fatty, and waxy casts; and microscopic
hematuria. Absorption of the lipid-containing proteins by the RTE cells followed by cellular sloughing produces the
characteristic oval fat bodies seen in the sediment examination. (Strasinger)

100. Xanthochromia is a term used to describe CSF supernatant that is pink, orange, or yellow. A variety of factors can
cause the appearance of xanthochromia, with the most common being the presence of RBC degradation products.
Depending on the amount of blood and the length of time it has been present, the color will vary from pink (very
slight amount of oxyhemoglobin) to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobin to
unconjugated bilirubin). Other causes of xanthochromia include elevated serum bilirubin, presence of the pigment
carotene, markedly increased protein concentrations, and melanoma pigment. Xanthochromia that is caused by
bilirubin due to immature liver function is also commonly seen in infants, particularly in those who are premature.
(Strasinger)

101. In general, the CSF contains protein fractions similar to those found in serum. As in serum, albumin makes up the
majority of CSF protein. But in contrast to serum, prealbumin is the second most prevalent fraction in CSF. The
alpha globulins include primarily haptoglobin and ceruloplasmin. Transferrin is the major beta globulin present; also,
a separate carbohydrate deficient transferrin fraction, referred to as tau, is seen in CSF and not in serum. gamma
globulin is primarily immunoglobulin G (IgG), with only a small amount of IgA. Immunoglobulin M (IgM), fibrinogen,
and beta lipoprotein are not found in normal CSF. (Strasinger)

102. A fresh semen specimen is clotted and should liquefy within 30 to 60 minutes after collection; therefore, recording
the time of collection is essential for evaluation of semen liquefaction. Analysis of the specimen cannot begin until
after liquefaction has occurred. (Strasinger)
 P a g e | 20

103. Measurement of the amount of hyaluronate polymerization can be performed using Ropes, or mucin clot, test.
When added to a solution of 2% to 5% acetic acid, normal synovial fluid forms a solid clot surrounded by clear fluid.
As the ability of the hyaluronate to polymerize decreases, the clot becomes less firm, and the surrounding fluid
increases in turbidity. The mucin clot test is reported in terms of good (solid clot), fair (soft clot), low (friable clot),
and poor (no clot).

104. SYNOVIAL FLUID: Clear fluids can usually be counted undiluted, but dilutions are necessary when fluids are turbid
or bloody. Traditional WBC diluting fluid cannot be used because it contains acetic acid that causes the
formation of mucin clots. Normal saline can be used as a diluent. If it is necessary to lyse the RBCs, hypotonic
saline (0.3%) or saline that contains saponin is a suitable diluent. Methylene blue added to the normal saline stains
the WBC nuclei, permitting separation of the RBCs and WBCs during counts performed on mixed specimens.
(Strasinger)

105. Tests for Fetal Well-Being and Maturity

Test
Bilirubin scan
Alpha-fetoprotein
Lecithin-sphingomyelin ratio
Amniostat-fetal lung maturity
Foam stability index
Microviscosity
Optical density 650 nm
Lamellar body count
Normal Values at Term Significance
A450 > 0.025 Hemolytic disease of the newborn
<2.0 MoM Neural tube disorders
2.0 Fetal lung maturity
Positive Fetal lung maturity/
phosphotidylglycerol
47 Fetal lung maturity
55 mg/g Fetal lung maturity
0.150 Fetal lung maturity
32,000/L Fetal lung maturity

106. AMNIOSTAT-FLM: The presence of another lung surface lipid, phosphatidyl glycerol, is also essential for adequate
lung maturity. The production of phosphatidylglycerol normally parallels that of lecithin, but its production is delayed
in cases of maternal diabetes. (Strasinger)

107. ACCURACY: Closeness of the measured result to the true value. (Strasinger)

108. PRECISION: Reproducibility of a test result. (Strasinger)

109. EXTERNAL QUALITY CONTROL: Commercial controls used to verify accuracy and reliability of patient test
results. (Strasinger)

110. INTERNAL QUALITY CONTROL: Electronic, internal, and procedural controls contained within the test system
that ensures the reliability of the test system. (Strasinger)