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systems for processing blood cultures.pptx
1. Systems for processing Blood
culture
Dr. Rasika Deshmukh
Fellow in Clinical Microbiology
Tata Medical Centre
2. Blood culture
Specimen of blood sampled through one
venipuncture (possibly divided into multiple
blood culture bottles) for the culture of micro-
organisms
3. Definitions
• Manual blood culture system :Blood culture
system that processes blood culture bottles
without the use of automated equipment
• Automated blood culture system :Blood
culture system that uses equipment (an
automated incubator) for incubation,
agitation, and monitoring of blood culture
bottles for microbial growth
4. Methods for blood culture
Advanced
Manual methods Lysis centrifugation
system
Automated and
computerized system
Conventional biphasic
bottle
•The Oxoid signal
blood culture
system
•BBL Septi-Chek
blood culture
system
•Wampole
Isostat/Isolater
Microbial system
•BacT/Alert 3D
system
•BD BACTEC
system
•Versa TREK
system
5. Different broths
broth Short description
Glucose broth useful in infective endocarditis
Tryptic Soy broth (TSB) General purpose medium, favors Pseudomonas species
(inhibits Neisseria and S. pneumoniae)
Thioglycolate Favors growth of anaerobes
Brain-Heart Infusion (BHI) General purpose medium, facilitates recovery of yeasts and
Gram-positive organisms
Columbia broth General purpose medium, favors growth of anaerobes
Supplemented peptone broth General purpose medium; superior to TSB for most common
pathogens in blood culture
Hypertonic medium (Brucella
broth)
increases recovery rates of some bacteria,
including Staphylococcus aureus, Escherichia coli, Candida
species
Bile broth favors growth of Salmonella spp
Mycobacterial Broth Middle brook 7H9 with SPS
Fungal broth favors growth of Fungii
6. Additives
Sodium-polyanethole sulfate (SPS) 0.025- 0.030%
• Anticoagulant;
• inhibits lysozyme,
• inactivates clinically achievable concentrations of
some aminoglycoside and polymyxin antibiotics
• inhibits parts of the complement cascade, and
inhibits phagocytosis
• promoting the recovery of Gram-positive cocci,
• decrease the recovery of Gram-negative bacteria.
• Inhibits Neisseria species, Peptostreptococcus
anaerobius, Moraxella catarrhalis, and Gardnerella
vaginalis
7. Additives
• Gelatin: Counteracts the inhibition of growth of bacterial
species by SPS in-vitro
• Yeast extract: Promotes bacterial growth
• Saponin Lytic agent: (used in lysis-centrifugation system);
improves recovery of Streptococcus species
• Hemin (X-factor): Promotes growth of fastidious organisms
such as Haemophilus influenzae and Neisseria species
• NAD (V-factor): Promotes growth of fastidious organisms
such as Haemophilus influenzae and Neisseria species
• Pyridoxine: Promotes growth of pyridoxine-dependent
organisms such as certain Streptococcus species
• Para-amino benzoic acid: Inhibits the effect of sulfonamide
antibiotics
• CysteineReducing agent :improves recovery of anaerobic
bacteria and Streptococcus pneumoniae
8. Additives
• Penicillinase: inactivates penicillin
• Charcoal/resins: inactivates antibiotics(when
charcoal is used, interfere with Gram staining and
reading )
• Osmotic stabilizers: sucrose, sorbose and
mannitol, for cell wall deficient bacteria , RBC’s are
lysed.
9. Traditional blood culture system :
• Blood culture system
• Broth are dispense in special flat blood culture
bottle of 100-120 ml capacity
• Fitted with a screw cap with a centrl hole giving
access to a rubber or neoprene washer seal
• An adequate space above broth ensures that
blood is not injected under undue pressure and
some air is still available for strict aerobes.
11. Visual Detection of bottles for
bacterial growth
• Turbidity,
• Hemolysis,
• Puff balls,
• Discrete colonies on the surfaces of sediment
RBC’s
• Gas production
• However, recoverable bacterial growth may
occurs before turbidity is evident
• So, it is important to make subculture from bottle
as a routine
12. A) pellicle formation on surface; (B) gas production; (C) turbidity (left bottle: no
growth; right bottle: turbidity); (D) puff balls.
13. The Oxoid signal blood culture system
• Single bottle blood culture
• Principle:
Growing and metabolizing bacteria produce
CO2>>pressure in bottle increase>>forces
liquid into the signal chamber
16. BBL Septi-Chek blood culture system
• Two-part culture system designed for rapid,
efficient and easy detection and presumptive
identification of pathogens from blood
• Biphasic agar slide system
• TSB or Columbia Broth, Brain Heart Infusion
Broth and Thioglycollate Broth
• Resins
17. BBL Septi-Chek blood culture system
• Trisurface paddle faced with chocolate,
MacConkey and malt strip agar
• 1st subculture after 4 to 6 hours of incubation
at 35° C (inverted and the upright) and then
regular reinoculation
18. Wampole Isostat/Isolater Microbial system (Alere)
• Alternative to blood culture
• Good for fastidious or slow growing
• Method of choice for dimorphic fungii,
Malassezia furfur and Legionella spp
• Reduction of mean recovery time
Organism Mean recovery
time
conventional
method
Mean recovery
time isolater
method
Yeast 4.9 days 2.12 days
H. capsulatum 24.14 days 8 days
20. Wampole Isostat/Isolater Microbial system (Alere)
Special tube contain Saponin (lysed RBC and
WBC)
7.5 -10 ml blood is added to tube , mixed
thoroughly by inverting many times
Tube is placed in angle centrifuge and spun
3000rpm for 15 min
Sediment is aspirated and subculture on
appropriate culture media
21. Wampole Isostat/Isolater Microbial system (Alere)
• Advantages
• Can be used as quantitative method
• CFU/ml can be calculate from volume of blood
processed and number of colonies grew on agar
surface
• Disadvantages
• 2 to 8 fold in increase in contamination rate
>>disinfect the working area and processed the
sample in vertical laminar hood
• Lysis solution is toxic to some microorganism
• Specimen must be processed with 8 hours of
collection
• Labor-intensive>>each sample need to process
individual
22. Automated systems
• Radiometric : Available from 1970
• No radiometric : available from mid 1980
• Three most commonly used system
Bact/ Alert(Biomerieux)
BACTEC 9240/9120/9050(BD biosciences)
TREK ESP system (Trek diagnostic systems )
23. • Its first automated non radiometeric system
which continuously monitor for growth in
culture.
• Provides optimal environment for recovery of
wide range of bacteria, yeasts and
mycobacteria
BacT/ALERT®
24. Types of modules
• Control Module: This module enables
up to 6 Incubation Modules to be
operated from one set of electronics
and integrated computer hardware.
• Incubator Module: Four
incubation drawers of 60 cells
giving a total capacity of 240 cells.
Each drawer has an independent
shaking mechanism, enabling
static or shaken cultures within
the same system.
25. Type of bact/alert bottles
Aerobic Culture
BactAlert FA Plus
BactAlert PA Plus
Anaerobic Media
BactAlert FN Plus
Mycobacterium Media
BactAlert MP
26. Working of BacT/ALERT system
• Media
• Bottles and CO2 sensor
• Colorimetric detector and instrument
• Computer analysis and detection algorithm
27. Media
• Designed to support growth and to ensure optimal CO2
production.
Content
• Soybean casein digestive broth
• Sodium polyanethol sulfonate
• Pyridoxal HCL
• Menadione
• Hemin
• Activated charcoal
• L-cysteine
• Carbohydrate
• Aminoacid
• Atmosphere of N2, O2, and CO2, under vacuum
28. Continued…..
• Adsorbent Polymeric Beads [APB] : Act as an
Antibiotic neutralizing Agent.
It Contains Two APBs
• APB 1: Gold Beads
• APB 2: Brown Beads
Advantages of these two APBs :
• APB contains numerous pores which increase the
surface area. More pores equate to more surface area
to bind antimicrobials. The gold colored APB bind with
non-polar antimicrobials to neutralize such as
vancomycin and others. The brown APB bind to
positively charged antimicrobials to neutralize such as
the aminoglycosides and others.
• Cysteine is used to neutralize Carbapenemase group of
antibiotics.
29. Bottles and CO2 sensor
• A CO2 sensor is bonded to the bottom of each bottle and is
separated from the broth medium by a semipermeable membrane
which is freely permeable to CO2.
• Carbon dioxide produced by growing organisms diffuses across the
membrane into the sensor and dissolves in the water,
• Generates hydrogen ions according to the following equation:
CO2 + HO * H2CO3 *-> H++ HCO3-
• As C02 is produced and dissolves in the water, the concentration of
hydrogen ions increases and the pH decreases.
• This causes the sensor to become
lighter green and eventually yellow,
which results in an increase of red light
reflected by the sensor.
30. Colorimetric detector and instrument.
• self-contained incubator (the temperature can be
adjusted between 35 and 37°C ± 0.5°C), shaker,
and detector.
• rock continuously at a rate of 60 rpm.
• Each well contains a colorimetric detector
• Reflected light>>>voltage signal proportional to the
intensity of the reflected light and the
concentration of C02 in the bottle.
• The instrument scans each well once
every 10 minutes
• voltage signals are digitized and
transmitted to computer for analysis.
31. Computer analysis and detection
algorithm.
• The data points are plotted as reflectance
units versus time and result in a growth curve.
• The algorithm for detection of growth is based
on an analysis of the rate of change of CO2
concentration in each bottle.
• Thus, the concentration of CO2 in each bottle
is compared with itself over time
39. QC in BacT/ALERT
PLAN FOR QC CHECK OF BACT/ALERT CULTURES BOTTLES
GROWTH QC
• Monthly and at time of new lot or same lot of culture bottle
receive
• ATCC strain – Staphylococcus aureus (ATCC-29213)
E.coli (ATCC-25922)
Mycobacterium tuberculosis(ATCC-25177)
• Growth check with solid plate culture and Vitek 2 system
STERILITY QC
• Monthly and at time of new lot or same lot of culture bottle
receive
• Uninoculated bottle
40. QC in BacT/ALERT
GROWTH PROMOTION TEST OF BACT/ALERT CULTURES BOTTLES
• To check quality of culture media of Bact/alert bottles by adding of
minimum volume and minimum concentration of bacterial
suspensions
• ATCC strain – Staphylococcus aureus (ATCC-29213)
E.coli (ATCC-25922)
Mycobacterium tuberculosis(ATCC-25177)
• 0.50 McFarland density >>>Serially dilute the culture suspension
>>> final dilution of 1:10000000.
• Inoculate 0.4 ml in adult and 0.2ml in pediatric bottle
• All aerobic organisms should be positive within 48 hrs of incubation
References: Bact/alert 3D user manual
41. BD BACTEC system
• Full automated computerized system
• Same as BacT/Alert system except that it used
fluorescent rather than spectral light to
detected changes in concentration of CO2
BACTEC System capacity
The BACTEC 9240 System 240
The BACTEC 9120 System 120
The BACTEC 9050 System 50
43. QC of BACTEC bottles
Growth QC
• inoculated with 1.0 mL of a 0.5 McFarland Standard of either
Escherichia coli or Staphylococcus aureus prepared from a
fresh 18 - 24 h culture.
• The inoculated vial should be detected as positive by the
instrument within 72 hours
Sterility QC
• uninoculated vial should be logged into the instrument and
tested
• The negative control vials should remain negative throughout
the entire testing protocol.
44. Versa TREK system
• Detects both gas production and gas
consumption
• Production of CO2 is measure manometrically
• Changes in concentration of H2 AND O2 in
addition to CO2
45.
46.
47.
48. BACT/ALERT BACTEC 240 VERSA TREK
CAPACITY 240 position 10
drawers
240 240 position 10
drawers
VOLTAGE(v) 100 V+/- 10% 100-117+/- 10% 110 V+/- 10%
HEIGHT/WIDTH/DEPT
Y(cm)
90/48.8/60.8 88.9 /63.5/86.4 103cm/132cm/78
WATTAGE(w) 640 --- 800
CLERANCE(cm) Front : 68.7
Rear : 1.3
Top : -
Side : 0
Front : 122
Rear : 11
Top : 61
Side : 11
OPERATING TEMP 59 F to 82 F 65 F to 89.5 F 59 F to 82 F
HUMIDTY 10-90% 10-90% 10-90%
WEIGHT 90 kg w/o bottles 187.5 w/o bottles 298 kg w/o bottles
LIS integration yes yes yes
Vibration free/Dust
surface/no direct
sunlight
yes yes yes
49. Conventional vs Automated blood
culture system
Conventional blood culture Automated blood culture
Less sensitive More sensitive
Slower time to positivity Faster time to positivity
Increase chances of contamination Less chances of contamination
More false positive Less false positive
Cheap Expensive
Daily once or twice checking for growth No need for checking manually
Tedious and labor intensive procedure Simple
No maintainance Expensive maintainance
No continuous supply of electricity continuous supply of electricity
Good for smaller workload and labs Expandable units ,good for large workload
and labs
50. Comparative studies
•failure to agitate Oxoid bottles
•Problem in growth media
•redox potential of the medium may have
reduced detection of obligate aerobic
microorganisms such as P. aeruginosa and
Candida species
•atmosphere in the bottle's headspace
(initially just evacuated with negative
pressure and, after attachment of the signal
device, room air).
•BACTEC is the faster detection of positive
cultures by the radiometric method
•Attractive points are its simplicity of design
without need for expensive instrumentation,
low contamination, and ease of recognizing
positive signals.
51. •The Oxoid Signal system was significantly better for
detecting oral anaerobes than the radiometric Bactec
460.
•Previous studies comparing Oxoid Signal with
radiometric Bactec showed that without agitation the
Oxoid Signal was significantly poorer for detecting
streptobut with agitation this difference disappeared.
53. • For aerobic cultures, the VersaTREK REDOX 1
system displayed the shortest TTD
• For anaerobic cultures, the BACTEC lytic/10
anaerobic/F system containing hemolysin
displayed the shortest TTD
• The status of H. influenzae growing in
BacT/Alert FN bottles can effectively reflect
the ability of this system to detect fastidious
bacteria
54. • Fifty clinical isolates of Candida > 1:100 dilution of each yeast
suspension>0.1 ml in inoculated in 3 bottles
• BacT detected 100% and the Bactec detected 90% using aerobic
broth
• BacT and the Bactec detected 100% using mycological broth
• BacT detected 70% and the Bactec detected 10% using anaerobic
media
• mean time to Candida growth detection in the BacT was faster
• missing out some candida is potientially fatal
55. •Paired aerobic and anaerobic VTI media were compared with
standard aerobic and anaerobic 3D media
•no overall difference in yield or in time to detection was
detected between the two systems
•significantly more streptococci and enterococci as a group
were detected by VTI
•significantly more microorganisms were detected by VTI for
patients receiving antimicrobial therapy.
•False-positive rates for aerobic and anaerobic bottles,
respectively, were 1.6% and 0.9% for VTI and 0.7% and 0.8%
for 3D
56. • simple method to identify microorganisms
from positive blood culture broth within the
time taken to perform a Gram stain
•unique combination of selective lysis, density
centrifugation, and IFS can rapidly identify the
most common microbial species present in
positive blood cultures.
•method is based on intrinsic fluorescence
spectroscopy (IFS) of whole cells
•classified 99.6% of unknown samples to the
Gram level, 99.3% to the family level, and
96.5% to the species level
• 0.8% of results were discordant at the
species level
•Faster identification of the etiologic agent
may benefit the clinical management of
sepsis.
57.
58. Special considerations
• Fastidious organism and endocarditis
• Prolong incubation beyond routine cutoff
• Some do not produce sufficient CO2. fail to
trigger the detector
• Useful maneuver is blindly stain with Acridine
orange after 7 days or the total duration of
incubation
• Acridine stain preferred over gram stain
>>more sensitive and examine quickly
60. Quality indicator Definition
Portion of blood cultures that showed growth
with positive pathogen(positivity rate )
No of blood cultures showing growth with a
pathogen /total no of blood culture bottle
Total number of blood cultures No of blood cultures/1000 patient days
Missed opportunities No of missed opportunities for blood culture
sampling as assessed by patient review file
Contamination rate No of contaminate blood culture bottle/ total
no of blood culture bottle
Volume Volume per blood culture bottle
Number and proportion of solitary blood
cultures
Adult blood culture bottle consist of one
blood culture bottle instead of two.
Needle to incubator time Time interval between blood culture sampling
to incubation
Time to detection of growth Time interval between incubation to
detection of growth
Gram stain accuracy Correlation between Gram stain results and
culture results
61. Quality indicator Definition
Turn around time Time interval from registration of sample in
the laboratory to reporting of results to
clinician
Quality of antibiotic susceptibility testing
report
Correct interpretation and reporting of raw
results
62. • Autoclaving: 121°C during at least 15 min
• longer cycles when a higher volume of liquids (e.g.,
more blood culture bottles)
• Monitoring the autoclave cycle by using chemical
indicators is often not sufficient, as they will only
show the presence of steam but do not give an
indication of the temperature inside the liquids
• validated before taken into use with the help of
temperature sensors (if available) and biological
indicators, preferably placed inside (non-
inoculated) blood culture bottles.
• Polycarbonate (PC) and polypropylene (PP) bottles
withstand Autoclaving
Waste Management
63. References
• Koneman’s color Atlas and textbook of diagnostic microbiology 7th edition
• Bact/alert 3D user manual
• BACTEC 240 user manual
• VERSA TREK user manual
• Ombelet S, Barbé B, Affolabi D, Ronat JB, Lompo P, Lunguya O, Jacobs J, Hardy L.
Best Practices of Blood Cultures in Low- and Middle-Income Countries. Front Med
(Lausanne). 2019 Jun 18;6:131. doi: 10.3389/fmed.2019.00131. PMID: 31275940;
PMCID: PMC6591475.
• Turan DB, Kuruoglu T, Gumus D, Kalayci F, Serefhanoglu K (2018) Evaluation of
Factors that may Cause False Positive Growth Signals in Blood Cultures-As the Word
'Factors' will Include Both Microbial and Patients as well as Others. Int J Clin Med
Microbiol 3: 137. doi: https://doi.org/10.15344/2456- 4028/2018/137
• Kumar M, Shergill SPS, Tandel K, Sahai K, Gupta RM. Direct antimicrobial
susceptibility testing from positive blood culture bottles in laboratories lacking
automated antimicrobial susceptibility testing systems. Med J Armed Forces India.
2019 Oct;75(4):450-457. doi: 10.1016/j.mjafi.2018.08.010. Epub 2018 Dec 20.
PMID: 31719741; PMCID: PMC6838483.
