BACT 211 (WEEK 4)

TOPIC 3: SPECIMEN COLLECTION, HANDLING, AND TRANSPORTATION

SPECIMEN REQUISITION
• Patient’s Name • Ordering of Physician
• Hospital Identification Number • Exact nature and source of specimen
• Age and Date of Birth • Diagnosis
• Sex • Current antimicrobial therapy
• Collection date and time

TIMING
• Specimens should be collected on the acute phase(early) of an illness (or within 2-3 days for viral
infections) and before the medicines.
SPECIMEN COLLECTION
• Needle aspirations> swabs
• The average time of specimen collection is 2 hours.
• 15 minutes for shorter time
• 24 hours if it has preservatives
Note:
• We should always use moistened swab with NSS. Dry swab is not recommended.
• If the organism can’t be move at 2 hours of time, use a holding medium preferably Stuart’s or
Amie’s medium. This medium only holds the organism and does not support growth.
• In some organisms such as N. gonorrhea, we use Dacron, Rayon swab, since it contains fatty acid
and liver.
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Abscess (Lesion, wound, pustule, ulcer)
Superficial Moistened Wipe area with < 2 hours 24 hours/RT BAP, CAP, MAC Add CAN if
aerobic swab sterile saline smear
with Stuart’s or 70% alcohol suggests
or Amie’s mixed-gram
result,
aspirate, if
possible, swab
along edges.
Deep Anaerobic Wipe area with < 2 hours 24 hours/RT BAP, CAP, MAC Wash any
tansporter sterile saline (anaerobic) granules and
or 70% alcohol emulsify in
saline; aspirate
or excise
tissue
Blood or Bone Marrow Aspirate
Blood or Bone Blood culture Antiseptic < 2 hours <2 hours/RT Blood culture Draw blood at
Marrow tube or technique with tube, BAP, CAP time of febrile
Aspirate vacutainer 70% alcohol episodes, draw
tube with SPS 2 sets, from
right and left
arm

Note: The General rule in Bacteriology is using 25 °C to 27 °C as room temperature

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 BACT 211 (WEEK 4)
0.025 % SPS
• Collects in the bone marrow
• It prevents clotting and holds the organism
Note: We should always check if the concentration is just right, otherwise, microorganisms such as
Neisseria, fungi, and anaerobic organisms will die.
Heparin
• Second Option
• This can also be used but may inhibit some gram positive and yeast.
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Other Body Fluids
Amniotic, Sterile screw Disinfect skin < 15 minutes <24 hours/4 Culture Need needle
abdominal, capped, with Iodine or bottles, BAP, aspiration; May
bile, joint, anaerobic povidone- °C- pericardial CAP, MAC, need to
pericardial, transporter or Iodine and other Thioglycolate concentrate by
pleural blood culture fluids for broth, CNA centrifugation
fungal culture or filtration;
stain culture
<24 hours/RT- sediment
everything
else
Bone Sterile screw Disinfect skin Immediately/RT Plate Asap BAP, CAP, MAC, May need to
capped with Iodine or Thio homogenize
povidone-
iodine

AMNIOCENTESIS
• Amniocentesis is a procedure used to obtain amniotic fluid from the uterus for testing or
treatment.
Note: Amniocentesis is done to remove amniotic fluid and cells from the uterus for testing or treatment.
Amniotic fluid surrounds and protects a baby during pregnancy.
PARACENTESIS
• Paracentesis is a procedure to drain fluid from the abdomen, which can cause pain and other
problems.
ARTHROCENTESIS
• A procedure done to remove the synovial fluid accumulated around the joints.
PERICARDIOCENTESIS
• Pericardiocentesis is a procedure that involves draining fluid from the pericardium, the sac around
the heart.
THORACENTESIS
• Thoracentesis is a procedure to remove fluid or air from the pleural space, the thin gap between
the lungs and the chest wall.
Body Fluids needs to concentrated
-bones may need to be homogenized
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Cerebrospinal Fluid (CSF)
CSF Moistened Wipe area with < 15 minutes 24 hours/37 BAP, CAP Consider rapid
aerobic swab sterile saline testing,
with Stuart’s or 70% alcohol °C except for consider blood
or Amie’s viruses -4 °C culture, Add
for 3 days Thioglycolate
broth if CSF
was collected
from shunt

Note(s):
• Ural Testing should be tested with 4 °C below for 3 days.
• Virus is an intracellular organism, meaning it can only be seen inside the cell.

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 BACT 211 (WEEK 4)
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Ear
Inner Ear Sterile screw- Clean ear canal < 2 hours 24 hours/RT BAP, CAP, MAC -
capped tube or with mild soap
anaerobic
transporter
Outer Ear Moistened Wipe away < 2 hours/RT 24 hours/4 °C BAP, CAP, MAC -
aerobic swab crust with
with Stuart’s sterile saline
or Amie’s
Eye
Conjunctiva Moistened - < 2 hours/RT 24 hours/RT BAP, CAP, MAC -
aerobic swab
with Stuart’s
or Amie’s
Aqueous/ Sterile screw Prepare eye < 15 mins/RT <24 hours/RT BAP, MAC, Some
Vitreous fluid capped for needle 7H10 anesthesia may
aspiration inhibitory to
some
organisms
Corneal Bedside Local < 15 mins/RT 24 hours/RT BHI 10%, BAP, -
Scrapings inoculation to anesthesia Incubated at CAP, and SDA
BHI 10% with
28 °C if Fungal antibiotics

Note: Corneal scraping is often used when there is suspicion of an eye infection, such conjunctivitis (pink
eye).
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
GI Tract
Gastric Biopsy Sterile screw Anesthesia < 1 hour/RT 24 hours/4 °C Skirrow, BAP, Perform rapid
capped BBA urease,
breathing test,
and antigen
test for H.
pylori
Stool/ Feces Clean leak- - < 1 hour/RT <24 hours/RT BAP, MAC, XLD, May use
proof if in holding HE, Campy, methylene blue
container or EMB stain to detect
Cary-Blair if <24 hours/RT media fecal leukocyte
more than 1 if in holding
hour media 24-48 hours/4 DO NOT:
°C patients
confined for
>3 days and
initially do not
have diarrhea

Note (s):
• Helicobacter pylori is an example of bacteria that over produces acid.
• For us to know if WBC is present in the stool, we should dye it with methylene blue.
• For patients who’s confined for 3 days, the often gets nosocomial diarrhea, cause by the
Clostridium difficile.
• For collecting stools, Bed pan is used instead of the bowl in the CR.
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Female Genital Tract
Cervix Moistened Remove mucus < 2 hours/RT 24 hours/RT BAP, CAP, MAC, -
aerobic swab before Thayer-Martin
with Stuart’s collection
or Amie’s
Vagina Moistened Collection 1 < 2 hours/RT 24 hours/RT BAP, CAP, -
aerobic swab hour after Thayer-Martin
with Stuart’s that urination
or Amie’s
Remove
Exudate
Urethra Moistened Remove < 2 hours/RT <24 hours/RT BAP, Thayer- -
aerobic swab Exudate Martin
with Stuart’s
or Amie’s or
JEMBEC

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 BACT 211 (WEEK 4)
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Male Genital Tract
Prostate Moistened Remove mucus < 2 hours/RT 24 hours/RT BAP, CAP, MAC, -
aerobic swab before Thayer-Martin,
with Stuart’s collection CNA
or Amie’s and
sterile screw-
capped tube
Urethra Moistened Remove < 2 hours/RT 24 hours/RT BAP, CAP, -
aerobic swab Exudate Thayer-Martin
with Stuart’s
or Amie’s or
JEMBEC

Note: JEMBEC is used for bacteria’s such as Neisseria gonorrheae

SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Hair, Nails, Skin
Hair, Nails, Clean, screw- If nails or < 72 hours/RT Indefinitely/RT SDA Hair: collect hair
Skin Scrapings capped tube skin, wipe with with intact
70% alcohol shaft
Nails: clippings
Skin: Scrape
skin at ledging
edge of lesion
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Respiratory Tract
Sputum sterile, screw- Have patient <2 hours/RT 24 hours/4 °C BAP, CAP, MAC, Do AFB
capped tube brush teeth Pseudomonas
then gargle cepacian agar
with water
Throat Swab Moistened - <2 hours/RT 24 hours/RT BAP Swab posterior
(Pharynx) aerobic swab pharynx and
with Stuart’s tonsils
or Amie’s
SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS
PREPARATION
Tissue
Tissue sterile, screw- Antiseptic <15 mins/RT 24 hours/RT BAP, CAP, MAC, May need to
capped tube tehcnique CNA, homogenize
Thioglycolate
broth Do not allow
specimen to dry
out, moisten
with sterile
distilled water
if not bloody

SPECIMEN CONTAINER PATIENT TRANSPORT STORAGE MEDIA COMMENTS

PREPARATION
Urine
Clean-voided sterile, screw- Clean with < 2 hours/RT 24 hours/4 °C BAP, MAC Plate
midstream capped tube soap and quantitatively
container water, retract 1:1000
labia or
May include foreskin, 1:100 if patient
preservative collect is female of
midstream childbearing
age with WBC
Straight Sterile, screw- Collect the < 2 hours/RT 24 hours/4 °C BAP, MAC Plate
catheter capped next 15 ml quantitatively
container 1:1000 or 1:100
Foley Catheter
is not
recommended
Suprapubic Sterile, screw- Needle < 2 hours/RT Plate ASAP BAP, MAC, Plate
Aspirate capped aspiration Anaerobic quantitatively
container or agars, 1:1000 or 1:100
anaerobic Thioglycolate
transporter broth

Note: We flame quantitatively those urine tissue from a burn.

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 BACT 211 (WEEK 4)
GROSS EXAM
• Need to verify the specimen and add in
diagnosis
DIRECT MICROSCOPIC:
1. Quality of specimen can be assessed
Ex: Sputum, <25 squamous epithelial cell/ LPO or
<10 squamous epithelial cell/HPO.
2. Clinical can be given early indication
3. Specimen workup can be compared on the
culture
Note: If an organism has to be shipped, we should
do triple packaging. The figure below is an • Ex: 3 Bacteria on a smear, 2 on an aerobic
example of triple packaging. culture
• Ex: 1 Bacteria on smear, 4 on an aerobic
culture
BASIC DYE VS ACIDIC DYE
BASIC DYE
• Commonly used- Cationic dyes with
positively charged groups that adhere to
negatively charge molecules like nucleic
acids and proteins
Example: Methylene blue, crystal violet, safranin,
and malachite green
ACIDIC DYE
SPECIMEN PRIORITY: TABLE 6.3 LEVELS OF • Anionic dyes with negatively charged
SPECIMEN PRIORITIZATION groups that bind to positively charge cell
structures.
LEVEL DESCRIPTION SPECIMENS Example: Eosin and acid fuchsin
1 Critical/ Amniotic fluid
Invasive Blood STAINS BASED ON COMPLEXITY
Brain
Cerebrospinal fluid
Heart valves 1. SIMPLE STAINING
Pericardial fluid
2 Unpreserved Body fluids (not • Single stain is used- Directed towards
coloring the forms and shape of the cells.
listed for level 1) Example: Methylene blue
Bone
Drainage from
wounds
Feces
Sputum
Tissue
3 Quantitation Catheter tip
required Urine
Tissue for 2. DIFFERENTIAL STAINING
quantitation
4 Preserved Feces in • Divide bacteria into separate groups
preservative
Urine in • Directed towards coloring the components
preservative of the elements present.
Swabs in holding Example: Gram staining and Acid-fast bacilli (AFB)
medium (anaerobic
and aerobic) staining

HOW DO WE LABEL SPECIMENS/ BLOOD TUBE?

-Same as bacteriology
• patient’s first and last names;
• identification number;
• date;
• time (as required, e.g. therapeutic drug
monitoring); and
• identification of the person collecting
the specimen.
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 BACT 211 (WEEK 4)
GRAM STAIN • Baumgarten method- differentiate
Mycobacterium leprae from
Mycobacterium tuberculosis.
• Auramine-rhodamine method- selective for
the cell wall of AFB
3. NEGATIVE STAINING
• Demonstrate presence of diffuse capsule
surrounding some bacteria
• Excellent technique for studying bacterial
gas vacuole and viral morphology
• Appearance: bacteria as light-colored
bodies against dark background
• Example: India ink or Nigrosin dye
Note: In this type of staining, we only color the
background, hence why the bacterial fungi appear
transparent.
4. SPECIAL STAINING
STAINING TECHNIQUE CELLULAR
STRUCTURE/BACTERIA
Dyar Cell Wall
Anthony’s, Hiss and Capsule
Gin’s
RULE: All cocci are Gram positive except: Nigrosin Capsule
LETTER BACTERIA Neisser Metachromatic granules
No Neisseria Albert Metachromatic granules
Boyfriend Branhamella Domer Endospore
Muna Moraxella
Vilma Veillonella CULTIVATION
• There is a need to process first. Urine
RULE: All bacilli are Gram negative except: needs to be concentrated by
LETTER BACTERIA centrifugation.
B Bacillus
L Lactobacillus HOMOGENIZATION
L Listeria
A Actinomyces • Grinding or Mincing or Squash/Crush Prep
C Clostridium
C Corynebacterium SWAB HAS MANY TYPES
M Mycobacterium • If fiber: Vortex in 0.5-1 ml saline or broth
E Erysiphelothrix
N Nocardia for 10-20 seconds
TRADITIONAL FIBER SWABS
HEADS UP: • Yards or fiber are wrapped around an
applicator
• Removal of MgRNA
• Sample is trapped in swab fibers
• Aged, dying and autolyzing cells.
Old cells may lose their ability to retain HUAREE ® FLOCKED SWABS
stains
• Antibiotic-treated bacterial cells have • Nylon fibers applied to applicator using
atypical staining reaction Copan’s proprietary flocking process
• Using acidic iodine during staining • No inner core to trap sample
• Due to a technical error or the wrong use LOOPS MATTER IF:
of stains
• Urine or Tissue from burn victims; we
TYPES OF AF STAIN should plate quantitatively
• Ziel-Neelsen/Hot staining Method CULTURE MEDIA
• Composed of a mixture of nutrients such
• Kinyoun’s Cold staining Method as carbon, nitrogen, sulfur, phosphorus,
hydrogen, oxygen and buffers
• Pappenheim method- differentiate • Types:
Mycobacterium spegmatis from
Mycobacterium stuberculosis. • Liquid
• Semi-solid
• Solid Medium
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 BACT 211 (WEEK 4)
TYPES OF CULTURE
• PURE CULTURE- It is composed of only one
species
• MIXED CULTURE- It is composed of more
than one species
• STOCK CULTURE- It is composed of
several culture species contained in a
separate culture medium (one species per
culture medium). It is used for academic
and industrial purposes.
ACCORDING TO CONSISTENCY
1. LIQUID MEDIUM
• It does not contain any amount of agar
• Also called broth
• Nutrients are dissolved in water
• Turbidity is measured
• It allows the growth of aerobes,
anaerobes, and facultative anaerobes
• Example: Brain heart infusion, trypticase
soy broth (TSB), and thioglycolate broth.

2. SEMI-SOLID MEDIUM
• It contains 0.5% to 1% Agar 2. NON-SYNTHETIC OR COMPLEX MEDIUM
• It is used to observe bacterial motility • Medium in which some substances are
And detect indole and sulfide production known
• Ex: Sulfide Indole Motility (SIM) Medium • Peptones, Meat and Yeast Extract
➔ Anaerobes- bottom part • Useful for isolation of medically
significant bacteria
➔ Aerobes- upper layer
• Ex: Nutrient broth (NB) broth medium,
3. SOLID MEDIUM TSB and MAC agar.
• It contains 2% to 3% agarose 3. TISSUE CULTURE MEDIUM
➔ Melts at ≥ 95 °C • Used for obligate intracellular
➔ Solidifies below 50 °C (now called agar) bacteria (Rickettsia and Chlamydia)
• Example: W138 cells, HeLA 299 Cells
• Ex: Triple sugar iron (TSI) agar, and McCoy cells.
MacConkey (MAC) agar, Blood agar plate HeLa 299 cells Human Cervical
(BAP), and Chocolate agar plate (CAP) Tissue
McCoy cells Fibroblast
W138 cells Fibroblast
ACCORDING TO COMPOSITION
ACCORDING TO DISPENSING OR DISTRIBUTION
1. SYNTHETIC OR DEFINED MEDIUM 1. Plate Media- Distributed into the
• Medium in which all the components are dish or plate
known 2. Tube Media- Prepared as either
• Used for research purposed liquid, slant, butt and slant or butt
• Preferred for isolation of cyanobacterium ➔ Examples:
and chemoorganotrophs ➔ Triple sugar iron (TSI)
• Ex: BG-11 medium ➔ SIM- Simmon’s citrate agar
➔ Lysine iron agar (LIA)

ACCORDING TO USE
1. NUTRITIVE MEDIA
• Routinely used in the laboratory and
without additional supplement
• Support the growth of most non-
fastidious bacteria
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 BACT 211 (WEEK 4)
• Non-selective ➔ Colistin-Nalidixic acid (CAN) agar; Gram
• Usually composed of meat and soybean positive bacteria.
extract
• Ex: Nutrient agar, Nutrient broth, and WHAT SHOULD WE ADD TO BE SELECTIVE?
Tryptone soy broth (TSB), Blood Agar To prevent growth of ADD:
Gram Positive Crystal/ Gentian
Plate (BAP), Chocolate Agar Palte (CAP) Bacteria
Violet, basic/carbol
fuchsin and bile salt
2. ENRICHMENT MEDIA (LIQUID-TYPE MEDIA) Gram Negative Potassium tellurite
• Also called back-up broth Bacteria and sodium azide
For Swarming Alcohol and chloral
• Also called supplemental broth Bacteria hydrate
• Allows detection of:
➔ Small numbers of organism present THE MEDICALLY IMPORTANT CULTURE MEDIA
may be detected
➔ Organism damaged by the 1. BRAIN-HEART INFUSION
antimicrobial COMPOSITION PURPOSE REMARKS
Peptone Bacterial Can be broth
➔ Detection of anaerobes in aerobic identification or agar
culture Phosphate
• Contain specific nutrients and without buffer Antimicrobial Can be with
additional supplement susceptibility blood or
Small conc of testing without blood
• Example: Alkaline peptone water, Selenite dextrose
F, Thioglycollate, Tetrathionate, Gram-
negative (GN) broth and Lim broth.

3. DIFFERENTIAL MEDIA
• These media allow the visualization of
metabolic differences between groups of
bacteria
• Example: MAC, BAP, eosin methylene blue
(EMB), and Hektoen Enteric Agar (HEA)

2. CHOCOLATE AGAR
COMPOSITION PURPOSE REMARKS
BAP+ hemin/ Neisseria Same as blood
x factor and gonorrhea agar except
nicotinamide Haemophilus that red cells
adenine are lysed to
Dunucleotide/
spp. release
hemin/x
NAD/V Factor factor and
nicotinamide
4. SELECTIVE MEDIA adenine
dinucleotide
• These media are incorporated with /NAD/ V
antibiotics, dyes, or chemicals to inhibit Factor
the growth of other organisms.
Examples: HEA, MAC, Xylose lysine
desoxychocolate (XLD) agar, Bismuth sulfate
agar (BSA), Mannitol Salt Agar (MSA), and
Thayer- Martin Agar (TMA).

Other selective media:

➔ Gentamicin blood agar: Streptococcus
➔ Bacitracin chocolate agar: Haemophilus
➔ Blood agar plate with ampicillin:
Aeromonas
➔ Phenylethyl alcohol: Gram positive bacteria

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 BACT 211 (WEEK 4)
3. COLUMBIA CAN WITH BLOOD
COMPOSITION PURPOSE REMARKS
Three peptone Growth of -
sources gram-
positive
5% bacteria
defibrinated
blood
Colistin and
nalidixic acid -
to inhibit
most gram
negative.

6. Sheep Blood Agar

COMPOSITION PURPOSE REMARKS
Protein Supports Detects
source: growth of all hemolytic
tryptones bacteria patters
Soy bean Also called
protein digest BAP
Sodium Rabbit or
chloride horse blood
4. HEKTOEN- ENTERIC AGAR may be used,
COMPOSITION PURPOSE REMARKS Sheep blood but the
Bile salts Growth of Slows the hemolytic
Salmonella and growth of patterns may
pH indicator; Shigella spp. - non- be
bromothymol blue green pathogenic inconsistent
blue and gram- with those on
negative sheep blood
acid fuchsin enteric
dyes bacteria
Also
differential –
non-SS is
orange to
salmon
colored
colonies

7. Modified Thayer-Martin
COMPOSITION PURPOSE REMARKS
Colistin- Supports Has
inhibit gram growth of N. modifications
negative gonorrheae such as
and N. additions of
Vancomycin- meningitidis trimethoprim
inhibit gram to inhibit
positive Proteus
organisms
Nystatin-
yeast
5. MacConkey Agar
COMPOSITION PURPOSE REMARKS
Crystal For Lactose
violet- inhibit differentiation fermenter-
Gram positive of gram- pink
and fungi negative
enteric lactose Non-lactose
Sugar: fermenters fermenter-
lactosepH and non- colorless
indicator: lactose
neutral red fermenters

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 BACT 211 (WEEK 4)

5. SPECIAL MEDIA
• Used to isolate bacteria with specific
growth requirements
Example: Lowenstein- Jensen (LJ) medium and
Thiosulfate citrate-bile salts-sucrose (TCBS)
agar.

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