1. The future of Laboratory Microbiology (and in particular bacteriology)
This is a work in progress!

2. Overview Background/history Plate streakers Automated Urine Analysers
Blood cultures
Automated Susceptibility Testing
Automated ID testing
Malditof
TLA
Molecular assays in bacteriology
Automation and volume
Automation and commerce

3. Bacteriology: The Origins
1881-Nutrient Gelatin first demonstrated by Robert Koch
1887 agar plates developed
MacConkey agar first used
1900s Dyes used to make media selective
Blood Agar first used to study haemolysis of streptococci
1881 – Nutrient Gelatin was used by Robert Koch to grow bacteria on – previously bacteria had been grown in liquid media – with purity being ascertained by checking the microscopy. Obviously this wasn’t 100%.
So Koch decided to try for a solid media – and did use sliced potatoes – however in the end opted for making a liquid media as before – and adding gelatin to make it solid. From there we have our agar plates.
Then obviously once they figured out agar – they started adding things to it to inhibit or enrich certain bacteria.

4. So what's changed??, not much really
Agar plates are still the main media for the majority of microbial sub-culturing and the backbone of the bacteriology lab.
Disc diffusion is still used in the vast majority of laboratories – if not all to some degree
Automated ID/Sens machines (VITEK and PHOENIX) have improved – however many of the concepts are still the same, using broth dilution breakpoint methodology.
Development in molecular diagnostics.
So whats changed – Recently i heard someone say that if Louis Pasteur or Robert Koch walked into a Microbiology laboratory today – they would still recognise many of the tools of the trade and probably be able to sit down and read a bench. Obviously plenty of things have changed. But look at the main staples of microbiology
Agar plates
Disc diffusion
And yes even tho the automated ID/sens machines have gotten smaller and more efficient the concepts dreamed up in the 70’s are still there.

5. What about some other techniques?
Manual streaking of plates has not really changed since solid agar plates were first used
Microscopy is mostly unchanged –
Incubators while probably more reliable are essentially the same
Plate reading has not really changed over years – although you are not supposed to sniff plates anymore….but we know it happens
Even if we look at other things – I am sure they are not done all that differently now then they were 50 years ago.
Streaking of plates with a loop
Gram staining of direct samples and colonies
Chucking our bugs into nice big warm cupboards
Picking up a plate – casting our beady little microbiologist eyes over it – and taking that sneaky sniff

6. Pre-analytical – Plate Streakers
Select appropriate media
Loads the samples
Spreading the inoculum to obtain isolated single colonies following incubation
Suppliers: WASP (Copan) Previ-Isola (BioMerieux) Innova (BD) and Inoqula (KIESTRA)
Not all systems include Gram stain preparation
Recent Studies show that the number of isolated colonies with both the Previ-Isola and WASP system are higher than when inoculation is performed manually.
The makers of the machines say these machines give a more consistent and better quality of plate streaking – leading to less need for secondary replating to gain pure growth – and ultimately a faster average time to ID/SENs and at the end of the day that is what the clinicians are wanting from us – RESULTS and fast

7. Here we have the Previ-isola on your left and the Innova on the right
Here we have the Previ-isola on your left and the Innova on the right. Both can be loaded with different media and will take a range of samples

8. In this picture we have the Inoqula on the left and the WASP on the right.
As far as I could tell – none of these machines deals with the common swab as yet – a liquid based transport media is need for all the automated plate streakers. Please correct me if anyone knows different.

9. Automated Urine Analysers
Automated Dip-strip inoculation and reading
Cell counts performed automatically – either by flow cytometry or (more recently) high resolution optics taking pictures of cells
Present now in many labs
Lots of suppliers – eg Sysmex, BD Iris etc been around for a while now probably the most common piece of automation in a microbiology laboratory
Although – some now a almost completely hands off having the dipstrips loaded on board – requiring the user only to place the urine on the machine and bingo bango presto you have a dipstrip reading and cell count – not sure if they culture the urine for you yet??

10. Blood Cultures Standard in most diagnostic labs, varying sizes.
Better detection times
More advanced media
Reduced total incubation before calling a bottle a final negative
Suppliers: BacT/Alert (BioMerieux) BACTEC FX (BD)
Actually this is probably the most common piece of automation in a micro lab....from bench top units that take 20 or so bottles – to the big beasts which hold plenty more.

11. Biomeriux lastest Bact/Alert on the left and the lastest BD FX on the right

12. The Automation of Susceptibility Testing
1940s started playing with putting antimicrobial agents into agar, both for selective culture and susceptibility testing.
Late 1940s: diffusion techniques on filter paper, 6mm disc 1947
1966 Kirby/Bauer attempt to standardise with their disc diffusion technique
1975 This technique becomes the basis for the NCCLS (CLSI) standards.
We all know about Alexander Flemming and his penicillin.
But disc diffusion techniques were trying to be standardised by the 1960’s and by 1975 the NCCLS had made the Kirby/Bauer method its standard for its interpretations. And they still use it today.

13. The Automation of Susceptibility Testing
1974 – First automated AST marketed by Pfizer called the Autobac
1977 Abbott introduce the MS 2 System
1977 McDonnell Douglas Corporation launch the AMS System – this was the predecessor to the first Vitek Machine
1977 First standarised microtitre plates with Antibiotics introduced – leads the way for Microscan, Sensititre and BBL Spector
Hard to believe that automataed AST was around in the 1970’s
Anyone here familiar with the Autobac??
With the Autobac I (General Diagnostics, Morris Plains,
N.J.), four or more colonies were suspended in Autobac
inoculation standardization fluid, and the turbidity was standardized
with the Autobac nephelometer. Samples (2 ml)
were then inoculated into Eugonic (EUG) and into Low
Thymidine Eugonic (LTE) broth. Paired cuvette cartridges
were filled with the inoculated broths and then incubated
simultaneously in the same Autobac incubator. After 3 h,
growth indices and light scattering indices (LSIs) were
determined as recommended by the manufacturer. A high
LSI indicates that the organism being tested is susceptible to
the antibiotic.
With the MS-2 (Abbott Laboratories, Irving, Tex.), four
or more isolated colonies were suspended in sterile distilled
water, and the suspension was adjusted to the turbidity of a
0.5 McFarland standard with the API-IR. Cuvette cartridges
were inoculated and incubated as recommended by the
manufacturer.
With the Microscan Microdilution MIC test panels (Microscan,
Inc., Campbell, Calif.), portions of several colonies
were inoculated into MIC test panels as recommended by
the manufacturer. Trays were incubated at 35°C and read at
18, 24, and 48 h. As recommended by the manufacturer, S.
aureus ATCC was used as the quality control strain in
Microscan test panels.
Isolates were also tested in the API 3600S (Analytab
Products, Plainview, N.Y.) by methods recommended by
the manufacturer. Inoculated rotors were incubated at 35°C
and read at 18, 24, and 48 h.
RESULTS

14. Automated ID and Sens Automated ID testing available since 1977
Biochemical substrates miniaturised and read by colourmetric or fluormetric means
Available in many bigger labs
Multiple Antibiotics in different dilutions available on Cards or panels to ascertain MIC – Expert Functions
Suppliers: Microscan Walkaway (Dade Behring) Vitek2 (BioMerieux) BD Phoenix (BD)
Cost neutral on ID part but not on susceptibility testing. What will happen to these systems with introduction of Maldi-tof?
As I mentioned before these have actually been around a while – although they have become more sophistocated over the years.
They provide – just like all automated equipment – standardised results that are not affected by individual human error in the reading – and while they may not be perfect and certainly none of them are – they do deal with probably 80% of a laboratories workload very efficiently and with little fuss

15. Here we have the beasts – BD Phoenix on the left – Microscan walkaway on the right and the Vitek2 down the bottom – automation is moving into the preperation of the specimens to be tested in these machines. The Vitek2 dilutes the specimens, vacuum fills them and seals the cards – reducing the time spent one each spec. The BD Phoenix has also introduced a similar system that reduces the amount of hands on time preparing the specimen.

16. MALDI-TOF Protein based spectral identification of bacteria
Identifications available in literally minutes – not hours
Tiny amount of bacterial growth needed – not affected by media or incubation conditions
Minimal cost per test, virtually no consumables
Suppliers : BD/Bruker, BioMerieux
This is the really exciting stuff.
Up until now most automation has been replacing exactly what a human would do with a mechanical analog.....but MADLI-TOF turns microbiology upside down and shakes it.
Matrix Assited Laser Deabsorption Ioniaztion – Time of Flight
This is COOL
You add a toothpick smear of a bacterial colony to a tiny circle on a detection plate...add a matrix – let it dry and chuck it on the machine.
The machine zaps that tiny circle that is full of bacteria encased in a crystalline matrix and it zaps it a lot. With every zap the proteins are released and fly up a flight tube – the size of the molecule decides how quickly it gets up the tube and you get a spectra for every zap....over 100 zaps produces over 100 spectra – these spectra a sent to a database of known spectra and using advanced alogrithms they whip back the most likely answer.
This is done in minutes – Myself Chris and Kay (and Michael) had the privilage to see one of these in action last week...and it does everything exactly as I have said here. Now...it isn’t always going to give you and ID – and sometimes it gets it wrong – but it doesn’t get it wrong anymore than the techniques we use now – in fact it gets it wrong less in most instances – and it happens so quick that its almost feels like a magic trick – i certainly expected a rabbit to pop out of the top of the machine.
The other advantage is the cost per test – less than a dollar per test – as consumables = The Matrix and.....thats it.

18. Bruker have teamed up with BD and have the Microflex here on the left – this is the machine we saw last week. And Biomerieux have the Vitek MS here on the right.

19. What is coming? What is next in the world of Microbiology Automation??
Some companies are in the process of producing TLA for Microbiology – similar to those seen in Biochem/Haem.
Putting specimens on a track – with no human intervention until plate reading time – and even then its not like you know it…
TLA for microbiology is not a thing for the future – companies are developing solutions right now – that could lead to a huge change in the way we do microbiology

20. This idea may seem a little far fetched – but when you look at front end automation in the core lab – you can see it isn’t really that crazy.

21. We all know what this is right??

22. Hands Off Microbiology!!
What happens if we no longer have to pick up plates.
Smart incubators that take pictures of all the plates inside it are being deveoped. With touch screen technology similar to that used in Ipads, it would be possible for a scientist to “swipe” his or her way thru the days plate reading.
What about testing I hear you cry!?!?!
Using that same touch screen technology scientists could zoom in on specific colonies

23. Spanner in the works….
All of this new automated technology assumes that bacterial culture on agar plates will remain the cornerstone of microbiological diagnosis for the forseeable future.
However molecular assays becoming more commonplace….even in bacteriology.
Virology now becoming increasingly molecular. Viral culture will eventually disappear from the diagnostic scene.

24. Some of the current molecular assays available commercially for bacteriology
Chlamydia + gono PCR
Illumigene (LAMP) for C.difficile toxin, Gp B strep
PCR for Group B strep and MRSA and VRE & ESBL enzymes
PCR Bordetella and C.difficile toxin
PCR and gene probe for mycobacteria
DNA probes for enteric specimens.
DNA probes for candida/BV/trichomonas
16sRNA identification

25. What is in the pipeline for molecular assays in bacteriology?
Microarrays
Still in research phase.
Still need extraction and amplification step.

26. Why is bacteriology not completely molecular?
Cost: Cost of Culture even including labour is inexpensive.
Culture allows Quantification of bacteria (but does it matter that much really?)
Looking for a range of different pathogens. Current methods aren’t that good at it. PCR gives up at about 5.

27. Will the agar plate eventually become defunct?
Yes, but possibly not in my working lifetime.
The companies producing culture automation are not daft. If they thought that agar plates would disappear in the next ten years, then they would not be R&Ding plate streakers, smart incubators etc.
Leasing, not buying outright such equipment would be prudent.

28. Automation and Volume
Automated methods most cost-effective when handling large volumes of samples.
Favours larger laboratories.
Interfacing is key
Most of the cost savings are in labour.

29. Automation and Commerce
Two main market players currently in this field, BD and Biomeurieux.
As the number of pieces of automated equipment in a lab increases, there is more and more incentive for laboratories to “affiliate” with a company for all their automation requirements.
Interesting times ahead….