Myths – pharmaceutical microbiology

Dr. Tim Sandle

Introduction - 7 myths1. Colony Forming Units – what are they?2. Microbiology laboratory cabinets – always work?3. Media growth promotion – can it be skipped?4. Microbial distribution in cleanrooms – free

floating?5. Environmental monitoring parameters – can

they be pre-set?6. Bunsen burners needed to create aseptic space–

or not?7. Identification results– always believable?

MythsWhat is a myth?

Myth ~ a traditional or legendary story with or without a determinable basis of fact or a natural explanation.

Myths around the ‘Colony Forming Unit’

Myth – CFU’s tells me how many bacteria there are? #1Not always:

Traditional culture based microbiological methods are variable,

Plate counts are an approximation of what is present,Many microorganisms will not grow on standard media

or their physiological state does not promote recovery,Dilution errors lead to poor recovery e.g.:

Over dilution, Under dilution = confluent growth

Aim of the ‘countable range’ cf Sutton “Accuracy of Plate Counts”, Journal of Validation Technology, 17 (3): 42-46

Counting errors can occur

Myth – CFU’s tells me how many bacteria there are? #2Often a CFU is not a

single bacteriumA colony could arise

from one cell or several.

Issue can occur through: Poor sample mixing e.g.

bacteria clumping together,

Poor plate mixing, Settle plate picking up

skin detritus.

Myth – sampling from anywhere within a colony is equalWith pure colonies, cells

experience different local conditions:Near the middle of the

colony, cells starve for nutrients, and accumulate wastes,

Cells in the middle of the colony are in stationary phase,

Leading edge cells are in log phase,

Mutations can occur - genetic diversity.

Myth – Clean air devices are secure and contamination free

Myth – microbiological workstations always are laminarAre they unidirectional?

Only do when they are empty.

Materials and equipment disrupt air flow and cause the air to swirl.

This can spread bacteria across surfaces or to other objects in the hood.

To avoid contamination, clutter must be minimized.

Aseptic technique

Myth - Isolators never leakIsolators

Aseptic manufacturingCompoundingSterility testing

LeakageLoss of air

Leaks:Isolators leak a given

amount of their volume per hour.

Gloves are a vulnerable point.

Myth – media growth promotion is not necessary

Myth – let the manufacturer perform media growth promotion testing #1Vendor:

Challenges lots plate media with a type culture from a culture collection

Uses a low level challenge (< 100 CFU)

Tests against previously released media Compare growth rates

Myth – let the manufacturer perform media growth promotion testing #2In-house testing:

Good practice to consider environmental isolates.

There can be a case for reduced testing, but:Need to verify the supplierNeed to account for different temperatures of

useNeed to consider if all appropriate control

strains are includedTransport issues

Heat shock

Myth – microbes in cleanrooms are ‘free floating’

Myth – microorganisms are free floating #1Microorganisms in

cleanrooms are rarely ‘free floating’Most are found on skin

flakes shed by operators.Or attached to dustTypical number (Whyte)

= 4 organisms. Argument for assessing

particles >0.5 µm in size. Argument for positioning

settle plates inside UDAFs.

Myth – microorganisms are free floating #2Microorganisms in air

Do not grow, air is not a natural biotope. Die off:

Relative humidity Lack of oxygen UV light

Those attached to water droplets can survive, potentially grow and travel long distances. Travel through passive

movement

Myth – There are ‘universal’ conditions for environmental monitoring

Universal conditions for environmental monitoring #1Do “universal conditions” for environmental monitoring

exist?Issues:

Not all microorganisms are culturable;Those that are culturable will not grow on all types of

media; Those that are physiologically weak (‘stressed’) will take

longer to grow than others;Our ‘microbiome’ is more complex than previously thought,Environmental monitoring methods are limited in

meteorology and variable in application.Therefore, we cannot expect to capture or to grow

everything but we need a standard set of conditions.

Universal conditions for environmental monitoring #2Some decisions required:Whether to select?

A general medium incubated across suitable temperature range, or

Two media – typically ‘bacterial’ and ‘fungal’,

Consideration of periodic selective agar / incubation conditions use.

Once agar has been selected, establish appropriate incubation times.

References: Sandle, T., Skinner, K. and

Yeandle, E. (2013). Optimal conditions for the recovery of bioburden from pharmaceutical processes: a case study, European Journal of Parenteral and Pharmaceutical Sciences, 18 (3): 84-91

Sandle, T. (2014) Examination of the Order of Incubation for the Recovery of Bacteria and Fungi from Pharmaceutical Cleanrooms, International Journal of Pharmaceutical Compounding, 18 (3): 242 – 247

Universal conditions for environmental monitoring #3How much does this matter?

Accept the limitations,Aim for optimal recovery,Be consistent:

Locations of monitoring, Frequencies of monitoring, Times of monitoring, Cleanroom conditions for monitoring.

Myth – Bunsen Burners are needed to create aseptic space

Myth – Bacteria don’t lieIs it best not to "flame the

mouth of the flask" when transferring fluids, or when pouring autoclaved media into petri plates?Can increase the risk

through generation of aerosols /air current contamination transfer

Best technique: Rapid transfer, Holding the flask or tube

horizontal to avoid dust settling.;

Use single-use sterile disposable items.

Myth - Microbiological identification is infallible

Myth – if controls work, the ID is soundGram-stain

Easy to get a mixed colony, Old colonies lean towards

Gram-positives, Over decolorisation can occur, Bacillus species can appear

Gram-negative.Automated systems

Phenotypic systems are affected by phenotypic changes,

All systems are only as good as their databases,

Cross-contamination can occur.

Myth – if I’ve found organism x it must be xQuestion the result of the identification

Is it expected from the sample source?Have I really got Bacillus anthracis? Or

Prochlorococcus spp.? Or Thermus brockianus? Most identification systems work on the basis

of matching and probabilityMixed cultures produce odd results

Summary1. Colony Forming Units – what

are they?2. Microbiology laboratory

cabinets – always work?3. Media growth promotion –

can it be skipped?4. Microbial distribution in

cleanrooms – free floating?5. Environmental monitoring

parameters – can they be pre-set?

6. Bunsen burners needed to create aseptic space– or not?

7. Identification results– always believable?

Thank youPharmaceutical microbiology: http://www.pharmamicroresources.com/