Microbiology

1. CULTURE OF NORMALLY
STERILE FLUID
PRESENTER: BMLS GROUP 3
MUHAS, 2023
MICRO SPECIMEN PROCESSING

2. Introduction
Urine Processing and Culture
Cerebrospinal Fluid Processing and Culture
Blood Culture
Outline

3. The culture of normally sterile body fluids refers to the microbiological
examination of bodily fluids that are typically free from microorganisms
under normal conditions. These fluids include blood, cerebrospinal fluid (CSF),
synovial fluid, peritoneal fluid, pleural fluid, and amniotic fluid.
In certain medical conditions or situations, these fluids can become
contaminated, leading to infections and other complications.
Sample of sterile body fluid collected for culture, it is done using aseptic
techniques. The sample is inoculated onto various culture media that support
the growth of diverse of microorganisms.
The plates or bottles are then incubated at the appropriate temperature and
conditions to promote microbial growth.
Introduction…

4. The presence of microorganisms in normally sterile body fluids, can suggest
an infection or other underlying medical condition.
Not all microorganisms isolated from normally sterile body fluids indicate
infection, as some may be part of the body's normal flora or contaminants
introduced during the collection process.
The culture of normally sterile body fluids plays a crucial role in diagnosing
and managing infections, ensuring appropriate treatment, and safeguarding
the health of individuals.
Introduction..

5. Urine culture refers to the procedures carried out in a clinical or diagnostic
laboratory to examine urine samples for the presence of bacteria or other
microorganisms. This process helps in the diagnosis and treatment of
urinary tract infections (UTIs) or other urinary system-related conditions.
A midstream clean-catch urine sample is typically collected to minimize
contamination. The urine sample is sent to the laboratory for analysis.
Indications:
Suspected urinary tract infection: Symptoms may include frequent
urination, urgency, burning sensation during urination, cloudy or foul-
smelling urine, or pelvic pain.
Monitoring treatment efficacy: Follow-up urine cultures can be
performed after antibiotic treatment to ensure eradication of the
infecting microorganism.
Urine Processing and Culture

6. Sample Collection: A clean-catch midstream urine sample is collected
from the patient. This is done to minimize contamination from the
external genital area.
Sample Processing: It is usually divided into two parts: one for
macroscopic, microscopic and biochemical examination and the other
for culture.
Microscopic Examination: Portion of the urine sample is examined
under a microscope to detect the presence of bacteria, white blood
cells, red blood cells, or other cellular elements. This examination can
provide preliminary information about the presence of infection or
other abnormalities.
Procedures

7. Culture and Sensitivity Testing: The other portion is cultured on agar plates
that promotes the growth of bacteria. The culture is incubated for a specific
period, usually 24hours, to allow any bacteria present in the sample to multiply
and form visible colonies.
Blood Agar:
MacConkey Agar:
CLED Agar: Cystine Lactose Electrolyte Deficient (CLED) agar is a selective
medium commonly used for urine culture
Sabouraud Dextrose Agar:
Procedures…

8. Procedures…

9. Colony Identification: Colonies that have grown on the culture plates
are examined. Different tests and techniques, such as Gram staining,
biochemical tests, are used to identify the types of bacteria present.
Antibiotic Sensitivity Testing: Performed to determine which
antibiotics are effective in treating the infection. This helps guide the
selection of appropriate antibiotics for treatment.
Reporting: The laboratory generates a report based on the findings of
the microscopic examination, culture, and sensitivity testing. The report
involve the type of bacteria present, the number of colony-forming units
(CFUs) per milliliter of urine, and the antibiotics to which the bacteria
are sensitive or resistant
Procedures…

10. Positive culture: The presence of a significant number of bacteria (usually
greater than 100,000 colony-forming units per milliliter) indicates a
urinary tract infection. The identified microorganism helps guide
antibiotic therapy.
Negative culture: A negative culture indicates the absence of significant
bacterial growth. However, it's important to note that negative cultures
do not rule out a urinary tract infection in all cases, especially in certain
clinical situations or when non-bacterial pathogens are suspected.
Contaminated culture: Contamination may occur if the urine sample is
improperly collected or contaminated during handling. The growth of
bacteria that are not typically associated with urinary tract infections
suggests contamination.
Interpretation

11. Several microorganisms can be commonly isolated from urine samples. The
most frequent ones are
Escherichia coli (E. coli).
Klebsiella pneumoniae.
Proteus mirabilis.
Enterococcus faecalis.
Staphylococcus saprophyticus.
Staphylococcus aureus.
Pseudomonas aeruginosa.
Enterobacter species: Enterobacter cloacae and Enterobacter aerogenes.
Candida species
Organisms

12. External contamination: Improper collection technique, inadequate
cleansing of the genital area, or collection from contaminated surfaces can
introduce bacteria into the urine sample.
Perineal flora: In some cases, bacteria from the perineal area can
contaminate the urine sample during collection, particularly in females.
Improper specimen handling: Prolonged storage or inadequate
refrigeration of the urine sample can lead to bacterial overgrowth and
contamination.
Contamination in Urine Culture

13. Cerebrospinal fluid (CSF) culture is a diagnostic test performed to identify
the presence of microorganisms in the cerebrospinal fluid surrounding the
brain and spinal cord. It is used to diagnose infections of the central nervous
system, such as meningitis or encephalitis.
Specimens are collected from: lumbar puncture or spinal tap, lateral
cervical puncture, citernal and ventricular cannula( shunt). In the laboratory,
the CSF sample is Macroscopically examined, then processed for
Biochemical test, Cell count and differentiation, protein, glucose levels, and
Culture.
 If only one tube of CSF is collected, submit to Microbiology first, otherwise
submit tube #2. Tubes should be numbered 1, 2, 3, with tube #1
representing the first portion of the sample collected.
Tube #2 or tube #3 are less likely to be contaminated by normal skin flora.
Cerebrospinal Fluid Culture

14. 1. Disinfect skin site with 2% tincture of iodine.
2. Insert needle with stylet at L3–L4, L4–L5, or L5–S1
interspace.
3. Upon reaching the subarachnoid space, remove the stylet
and collect 1–2 mL of fluid into each of 3 sterile CSF tubes.
4. Deliver tube #2 to Microbiology immediately.
 Collected volume of CFS at least 12mL, then partitioned into 3-
4 tubes, whereby;
Tube 1________________BIOCHEMISTRY
Tube 2________________MICROBIOLOGY
Tube 3________________CELL COUNT( HAEMATOLOGY)
Tube 4________________CYTOLOGY
 Refrigeration is not recommended for culture specimen as it
will kill fastidious bacteria.( N. meningitidis & H. influenzae)
Csf collection procedure –lumbar puncture

15. Hold sample against white paper and compare a
tube of distilled water. If any color observed reflect
abnormality.
Yellow color: xanthochromia( presence of bilirubin in
CSF)
Clear- normal
slightly turbid, cloudy, purulent-↑ pyogenic bacteria
and polymorphonuclear neutrophils (pus cells).
Contains blood –subarachnoid hemorrhage, due to a
traumatic (bloody) lumbar puncture
 Contains clots-webbed fibrin clot in MTb, and
traumatic fibrin clot
Macroscopic examination of csf

16. It is commonly done by using improved Neubaur chamber whereby total
cell count ( i.e., WBCs and RBCs) are counted.
Differential WBC count is done using Romanowsky( Leishman)stained
smear in which 60%-80% lymps, 30% monocytes and macrophages and 2%
other cells is considered normal
Biochemically, protein( 14-45mg/dL) and glucose( 40-80mg/dL) are
considered normal.
Cell count & biochemistry

17. Culture
Chocolate agar and blood agar- When Gram positive diplococci are
seen in the Gram smear, add an optochin disc to the blood agar plate to
assist in the identification of S. pneumoniae. Incubate in 5-10% CO2 for
48hrs
MacConkey Agar(if CSF is from newborn infant) and Saboraud Agar.
India ink preparation when cryptococcal meningitis is suspected
Wet preparation to detect motile amoeba( N. fowleri) or when no
bacteria seen in CSF with low glucose, high pus cells and high protein.
Wet preparation and Giemsa smear when trypanosomiasis
meningoencephalitis is suspected( pre treatment before sample
collection is recommended to prevent introduction of trypanosomes in
spinal cord)
culture of csf

18. Chocolate agar and blood agar cultures
oLook particularly for: N. meningitidis(oxidase positive) and S. pneumoniae-
sensitive to optochin(grow ON CA and BA), and H. influenzae (only
chocolate agar). Perform antimicrobial susceptibility tests as indicated and
Beta-lactamase test for H. influenzae .
MacConkey agar culture
oLook especially for bacteria that cause neonatal meningitis e.g., group B
streptococci(GBS-50%) and E. coli(20%)
Examine and report culture

19. In cerebrospinal fluid (CSF) cultures, microorganisms can be isolated in cases
of central nervous system (CNS) infections, such as bacterial meningitis or
fungal meningitis. Here are some commonly isolated microorganisms in CSF
cultures:
Bacterial pathogens:
Streptococcus pneumoniae
Neisseria meningitidis
Hemophilus influenzae
Group B Streptococcus (Streptococcus agalactiae)
Escherichia coli (in neonatal meningitis)
Listeria monocytogenes
CSF Culture..

20. Viral pathogens:
Enteroviruses (e.g.,
coxsackievirus, echovirus)
Herpes simplex virus (HSV)
Varicella-zoster virus (VZV)
Human herpesvirus 6 (HHV-6)
Epstein-Barr virus (EBV)
Cytomegalovirus (CMV)
Fungal pathogens:
Cryptococcus neoformans
Candida species (e.g.,
Candida albicans)
Aspergillus species
CSF Culture..
 Accurate diagnosis of CNS infections is crucial for initiating timely treatment
and improving patient outcomes.

21. Contamination in culture refers to the presence of microorganisms in a
specimen that are not reflective of a true infection
Skin flora: Contamination with skin microorganisms can occur during the
insertion of the needle during lumbar puncture.
Contaminated equipment: Improper sterilization of the equipment used
for CSF collection or processing can introduce bacteria into the CSF
sample.
Contamination during transport: Inadequate sealing or handling of the
CSF sample during transportation to the laboratory can lead to
contamination.
To minimize contamination in culture specimens, healthcare providers
should follow proper aseptic techniques during sample collection, including
thorough cleansing of the collection site and using sterile equipment.
Contamination in CSF culture

22. Contamination in culture refers to the presence of microorganisms in a
specimen that are not reflective of a true infection
Skin flora: Contamination with skin microorganisms can occur during the
insertion of the needle during lumbar puncture.
Contaminated equipment: Improper sterilization of the equipment used
for CSF collection or processing can introduce bacteria into the CSF
sample.
Contamination during transport: Inadequate sealing or handling of the
CSF sample during transportation to the laboratory can lead to
contamination.
To minimize contamination in culture specimens, healthcare providers
should follow proper aseptic techniques during sample collection, including
thorough cleansing of the collection site and using sterile equipment.
Contamination in CSF culture

23. A blood culture is a laboratory test in which blood, taken from the patient,
is inoculated into bottles containing appropriate culture media to determine
whether infection-causing microorganisms (bacteria or fungi) are present in
the patient’s bloodstream.
Aims of blood culture:
To confirm presence of microorganisms in the bloodstream
To identify the etiology of the bloodstream infection
To guide appropriate antimicrobial therapy by providing susceptibility
information of isolates
It involves collection of a blood sample from a patient, usually through
venipuncture. The collected blood is then placed in specialized culture bottles
containing growth media that support the growth of microorganisms.
Blood culture

24. After the blood sample is collected and placed in the culture bottles, the
bottles are incubated at appropriate temperatures usually at 37*C for a
specific duration, typically 24 to 48 hours.
 During this incubation period, if any microorganisms are present in the
blood, they will start to multiply and grow within the culture media.
 The culture bottles are checked for signs of microbial growth, such as
turbidity, color change or the presence of gas.
If growth is observed, Other laboratory techniques, such as Gram staining
and subculturing onto specific agar plates, are performed to identify the
type of microorganism present.
Blood Culture…

25. Blood cultures are typically drawn through venipuncture. Prior to the blood
draw, the top of each collection bottle is disinfected using an alcohol swab
to prevent contamination. The skin around the puncture site is then cleaned
and left to dry; some protocols recommend disinfection with an alcohol-
based antiseptic followed by either chlorhexidine or an iodine-based
preparation.
Sufficient blood volume is critical for the successful recovery of organisms
causing bloodstream infection. Recommended blood-to-broth-ratio is 1:5 to
1:10, commercial blood culture systems may use a smaller blood-to-broth
ratio (<1:5) due to the addition of sodium polyanetholesulfonate (SPS).
At least 2 tubes of blood should be collected.(aerobic and anaerobic
culture)
Blood Collection

26. For an adult, the recommended volume of blood to be obtained per
culture is 20-30 ml. Since each blood culture set includes an aerobic and
an anaerobic bottle, each bottle should be inoculated with approximately
10 ml of blood
For infants and children, the recommended blood volume should be
based on the weight of the patient, and an aerobic bottle should be used,
unless an anaerobic infection is suspected.
Specially formulated blood culture bottles are commercially available for
use in children <2 years of age. They are specifically designed to maintain
the usual blood-to-broth ratio (1:5 to 1:10) with smaller blood volumes,
and have shown to improve microbial recovery.
Blood collection

27. Bactec blood culture bottle
Blood culture bottle

28. Culture bottles contain a growth medium, which encourages
microorganisms to multiply, and an anticoagulant that prevents blood from
clotting.
Sodium polyanethol sulfonate (SPS) is the most commonly used
anticoagulant because it does not interfere with the growth of most
organisms.
The exact composition of the growth medium varies, but aerobic bottles
use a broth that is enriched with nutrients, such as brain-heart infusion or
trypticase soy broth and anaerobic bottles typically contain a reducing agent
such as thioglycolate. The empty space in an anaerobic bottle is filled with a
gas mixture that does not contain oxygen.
Blood culture bottle

29. Sodium polyanetholsulphonate (SPS)/ liquoid ,It has
Antiphagocytic,
Anticomplementary action. Inactivates the certain antibiotics like
gentamycin, kanamycin, streptomycin, polymyxin B.
It precipitates fibrinogen, B-lipoproteins, B 1C globulins.
 Other additives:
 Penicillinase- to inactivate penicillin
 Resin/charcoal- inactivates most antibiotics by adsorbing them to surface
of resin
 Osmotic stabilizer like sucrose, mannitol, sorbose- for cell wall
deficient bacteria, but RBC are lysed
 soybean-casein digest broth, yeast, amino acids, sugar, vitamins.
Blood culture bottle

30. Blood cultures should always be requested when a bloodstream infection
or sepsis is suspected. Some of the most common clinical symptoms in a
patient which may lead to a suspicion of a bloodstream infection are:
Undetermined fever ( ≥ 38°C) or hypothermia ( ≤ 36°C).
Severe local infections (meningitis, endocarditis, pneumonia,
pyelonephritis, intra-abdominal suppuration etc.)
Blood cultures should be collected:
As soon as possible after the onset of clinical symptoms;
Prior to the administration of antimicrobial therapy. If the patient is
already on antimicrobial therapy, recovery of microorganisms may be
increased by collecting blood sample immediately before administering
the next dose and by inoculating the blood into bottles containing
specialized antimicrobial neutralization media.
Indications of blood culture

31. The blood culture results can provide valuable information about the
susceptibility of the microorganism to different antibiotics, helping guide
the selection of the most effective treatment.
It is important to note that blood culture contamination is a common issue
that can lead to false-positive results. To minimize contamination, strict
aseptic techniques are followed during blood collection, and multiple sets
of blood cultures are usually obtained from different sites to ensure
accuracy and reliability.
Blood Culture..

32. Skin flora: The most common source of contamination in blood cultures
is the normal flora present on the patient's skin, which can be
introduced during venipuncture.
Inadequate skin preparation: Insufficient or improper disinfection of the
puncture site can increase the risk of contamination.
Contaminated equipment: Contamination can occur if the blood culture
collection equipment or culture bottles are not sterile or if they come
into contact with contaminated surfaces.
Contamination in blood culture

33. Common isolates;
Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas
aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae
Common contaminants;
Coagulase-negative staphylococci (e.g. Staphylococcus epidermidis group)
Streptococcus spp.
Pseudomonas aeruginosa and other Pseudomonas spp.
Corynebacterium (gram positive rods)
Propionibacterium acnes (anaerobic gram positive rods)
Coagulase-negative staphylococci are found to be contaminants 60-80% of
the bacteremia-causing bacteria
Microorganisms

34. Jawetz, Melnick and Aldelberg Medical microbiology and immunology:
27th edition.
Monica Cheesbrough District Laboratory Practice in Tropical Countries Part 2
Microbiology Manual
Online Resources
REFERENCES