This document discusses blood cultures and bacteremia/septicemia. It describes how blood cultures can help diagnose pyrexia of unknown origin and reveal pathogens to guide antibiotic therapy. Positive blood cultures indicate bacteria or toxins in the bloodstream (bacteremia or septicemia, respectively). The document provides guidelines for collecting blood culture samples, including volume based on patient age, timing of collections, and techniques to avoid contamination. It also discusses culture methods, common pathogens, and interpreting positive and negative results.

1. Bacteremia and Septicemia
Dr. Mahadi H Abdallah
PhD- Microbiology

2. Bacteriaemia and Septicaemia:
• Pyrexia of unknown origin (PUO):
Isolation of bacteria from blood with valuable:
1. Urgent need for antibacterial therapy.
2. Revealing the spp against therapy.
3. Providing a culture for the performance of in-vitro
drug sensitivity testing.
• Culture of specimen from local sites e.g. sputum, W.
Swab often yields a mixture of contaminating commensal
bacteria and potential pathogens 

3. Bacteriaemia and Septicaemia:
• Bacteriaemia: presence of bacteria in blood.
• Septicaemia: bacteria multiply and release toxins in blood
stream.
Blood Culture requested in two cases:
• Where possibility of septicaemia or bacteraemia is
suggested by present of fever, shock or their symptoms due
to local infection such as:
sepsis in surgical wound, puerperal sepsis, pneumonia,
meningitis, osteomyelitis or endocarditis.

4. Bacteriaemia and Septicaemia:
demonstrations of one in blood  significant, e.g.
pneumococci in sputum from suspected patient
with pneumonia it may be derive from site of
commensal carriage in the throat not from lung 
may be infection due to virus or Mycoplasma, but
when isolated from blood, it is pathogen in the
lung.

5. Contamination:
• Specimen contaminated with organism as
Staphylococci albus are the commonest
contaminant, but Diphtheroides bacilli, coliform,
Clostridium perfringens, Candida and other
organisms are rare.
• Patient with debility, immune deficiency

6. Collection of sample:
• Bacteriaemia  bacteria number depends on
number release from site of infection and removed
by R.E.S, so large volume of blood should be taken
10 - 20 ml.
• Endocarditis  6 samples at least interval of
several hours in course of 3-6 days.

7. Antibiotic in the blood:
Blood should be diluted 1: 5 or 1 in 10.
Advantage:
Reducing the concentration of natural antibacterial
constituents.
Reducing the conc. Of any therapeutic (antibiotic).
But when blood contains more than 20 times MIC,
Antibiotic prevent the growth.

8. Antibiotic in the blood:
So it's better to collect sample before use of
antibiotic. If used, sample must be collected after 2
hours after last dose at least or use antibiotic
inactivating, e.g. para-aminobenzoic acid 
Sulphanomide, broad spectrum B. lactimase 
Lactam antibiotic - 0.2 ml penicillinase  50 ml.

9. Culture systems:
Culture systems:
• Tradition System.
• Automated System.
Traditional method (Culture bottle):
Blood culture should be examined by gram stain film and
sub culturing on 2BA (1) aerobic with CO2 (5-10%).
(2) anaerobic H2/N2 with CO2 (5-10%). if meningococci or
haemophilus suspected CBA included.

10. Automated systems:
• These systems employ equipment that
automatically detects and early sign of bacterial
growth in special blood bottle. It depend on release
of radioactive CO2 (14CO2) into atmosphere of B-
bottle  degradation of 14C containing nutrients in
special medium.

11. Endocarditis:
• Sub acute infective endocarditis: S. viridians
• Acute endocarditis: S. aureus, S. pyogens,
Pneumococci.
• Liquiod (Na-polyanethol sulphonate). Anticoagulant
neutralized bactericidal subs polymyxin B,
streptomycin, kanamycin, gentamicn.

12. Blood culture

13. Blood cultureAim of the test
• An etiological diagnosis of bacteremia by aerobic and anaerobic
cultivation of the blood, with identification and susceptibility test of the
isolated organism(s).
• Blood culture should be made for cases with suspected septicemia,
endocarditis, and bacteremia secondary to localized infections
(pneumonia, intra abdominal abscesses, pyelonephritis, epiglottitis,
meningitis). In this case the blood culture may provide an etiological
diagnosis of the localized infection.
Types of specimen
• Whole blood.
Criteria of specimen rejection
• Blood collected in tubes or bottles other than aerobic and
anaerobic blood culture bottles.
• If the information on the label does not match that of the request form.
• Specimens for anaerobic blood culture received in aerobic bottles or vice
versa.

14. Common pathogens
Streptococcus spp Bacteroides fragilis and other anaerobic bacteria
Staphylococcus aureus Coagulase negative staphylococci
Listeria monocytogenes Enteric gram negative bacilli
Corynebacterium jeikeium Neisseria meningitides
Haemophilus influenza Non fermenter gram negative bacilli
Salmonella typhi Pseudomonas aeruginosa
Parasitic infection
Parasite can be found as transiently in the blood stream for example tachyzoites of Toxoplasma
gondii
Viruses
Epstein barr virus HIV virus
Cytomegalovirus Other human Retroviruses
Fungi
Candida albicans Cryptococcus neoformans
Other candida spp Coccidoides immitis
Histoplasma capsulatum

15. Bacteremia maybe transient, continuous, or intermittent.
The two major categories of blood stream infections are
intravascular those that originate within the cardiovascular system
and extravascular those that originate from bacteria entering the
blood circulation through the lymphatic system from another site of
infection.
Types of Bacteremia

16. Blood cultures should be drown prior to initiation of antimicrobial therapy,
if more than one culture is ordered the specimens should be drawn
separately at no less than 30 minutes apart to rule out the possibility of
transient bacteremia by self-manipulation by the patient of mucous
membrane in the mouth or by local irritation caused by scratching of the
skin.
The numbers of bacteria are generally higher in the acute, initial stage
than at a later stage of the disease, and small children usually have higher
numbers of bacteria in the blood than adults. The number is also higher
when the fever rises than when it is falling.
For patients expected to seed bacteria intermittently into the blood
80% of these are detected with the first culture and 99% within the three
cultures.
Specimen Collection

17. Before starting antibiotics therapy if time permits, its generally
recommended that the first two sets of blood cultures be taken one hour
apart and the third set after 3-6 hours.
Obtaining the blood culture one half hour before a temperature spike is
ideal because the highest concentration of organisms are circulating at that
time, because the temperature spike is usually un predictable an educated
guess must suffice in most cases when timing blood cultures.
Collection Time
Age of patient No. of blood bottle
Children below 2 years 1 mL of venous blood in 2 bottles
Children 2-5 years 2 mL of venous blood in 4 bottles
Children 6-10 years 3 mL of venous blood in 4 bottles
Children 11-15 years 5 mL of venous blood in 4 bottles
Children above 15 years and adults 5 mL venous blood in 3 sets of bottles (6 bottles).
Volume Of Blood Culture Collected Acoording To Age Of Patients

18. During blood culture collection all percussion should be taken to minimize the
percentage of contaminated blood culture, to reduce the chance of
contaminating organisms from the skin the vein puncture site should ideally be
prepared as follows;
I. Wash with soap, rinse with sterile water or saline.
II. Apply 1-2 % tincture of iodine or povidone –iodine and allow drying for 1-2
minutes.
III. Remove the iodine with 70 % alcohol wash, if the site again be palpated after
the iodine – alcohol preparation the finger must be disinfected or sterile
gloves worn.
A tourniquet is applied to the upper arm above the vein puncture site to
distend the anticubital veins.
Collection of Blood for Culturing

19. Remove Flip Caps from the tops of the selected culture
bottles. Disinfect the septa of the bottles with alcohol or iodine
preparation and allow to dry.
Perform venipuncture with syringe and collect the desired
amount of blood. If the vein is missed a new needle should be
used.
Transfer the recommended amount of blood into the culture
bottles using aseptic technique if desired. First fill the aerobic
bottle. Do not overfill the bottles! Any remaining blood may be
used for additional tests.
Label the bottles according to the routine procedure. When
using a sticker do not cover the tear-off section of the barcode
label .
Collection of Blood for Culturing
Note: 1:5 to 1:10 blood/broth ratio is the appropriate ratio to achieved, this dilution minimizes
the effects of microbial inhibitors present in blood and dilutes any antimicrobial agents.

20. The bottle incubated for 24 hour before plating to enhance the growth of
bacteria, aerobic bottle plate on blood agar, MacConky, and chocolate in CO2
incubator for 24 hour, anaerobic incubate anaerobically on blood agar for 48
hour, and the negative bottle should be reincubated and tested after 10 days
before discarded as negative culture. If slow growing organisms are
suspected as Brucella spp. its should be clearly indicated on the requisition
form and the culture bottles should be further incubated for 2-4 weeks before
being reported out as negative.
Specimen processing

21. Blood bottles
Trytic soy broth (TSB)
Pancreatic digest of casien.
Enzymatic soy digest.
Sodium chloride.
Dipotassium phosphate.
Dextrose.
Sodium polyanethol sulphonate(SPS)
Fluid thioglychollate medium (FTM)
Pancreatic digest of casien.
Enzymatic soy digest.
Sodium chloride.
Dipotassium phosphate.
Dextrose.
Sodium polyanethol sulphonate(SPS)
Sodium thioglychollate.
agar.

22. Sodium polyanethol sulphonate (SPS)
The anticoagulant in blood culture medium must not harm the
bacteria and must prevent clotting of the blood, which entrap bacteria
and prevent their detection .
The most commonly used preparation in blood media is 0.025% to
0.05% SPS.
In addition to it’s anticoagulant properities, SPS is also
anticomplementary, antiphagocytic, and interferes with the
activity of some antimicrobial agents.
(SPS)

23. Blood bottles
A set of blood culture: one aerobic bottle and one anaerobic bottle.

24. Blind Sub-Culturing syringe and drip methods

25. Blood bottles incubator

26. How to culture an intravenous catheter tips
When colonization of an indwelling
catheter is suspected of being the
focus for septicemia, the catheter tip
may be cutured to determine its
status.
After overnight incubation the
colonies are counted, A positive
culture result with greater than 5
CFU.

27. Interfering factors
Patient on antibiotic therapy
Result reporting
Any isolated organism will be reported. Antibiotic sensitivity will also be
included with the report.
Turn around time
Initial blood culture results will be reported as soon as it shows growth.
Final results with sensitivity will be issued after 24- 48 hours of the
initial report.
Negative results will be issued after 10 days of culture submission.
Post specimen processing

28. Interpretation of Positive Blood Cultures
Virtually any organism, including normal flora, can cause bacteremia.
A negative culture result does not necessarily rule out bacteremia;
false-negative results occur when pathogens fail to grow.
A positive culture result does not necessarily indicate bacteremia; false-
positive results occur when contaminants grow.
Gram-negative bacilli, anaerobes, and fungi should be considered
pathogens until proven otherwise.
The most difficult interpretation problem is to determine whether an
organism that is usually considered normal skin flora is a true pathogen.

29. Three negative sets of blood cultures in the absence of antimicrobial
therapy are usually sufficient to exclude the presence of bacteremia.
One set is seldom ever sufficient.
Prior antibiotic therapy may cause negative blood cultures or delayed
growth.
Blood cultures from patients suspected of having Brucella or Leptospira
must be requested as special cultures, Consultation with the laboratory
for special culture procedures for the recovery of these organisms prior
to collecting the specimen is recommended.
Yeast often are isolated from routine blood cultures. However, if yeast
or other fungi are specifically suspected, a separate fungal blood
culture should be drawn along with each of the routine blood culture
specimens.
Limitations

30. Mycobacterium avium complex (MAC) is frequently recovered from
blood of immunocompromised patients, particularly those with
acquired immunodeficiency syndrome, AIDS. Special procedures
are required for the recovery of these organisms.
Limitations continue ……
*Observed that performance of biphasic system to
be superior in recovering Brusella spp .The bi phasic
system is feasible and practical method , it has the
advantage of repeated exposure of agar medium to
actively proliferating organisms in the liquid broth
during sub culturing, which is simply by tilting the
bottle.
Castañeda Bi-phasic medium