Further methods of microbial identification

Biochemical methods (e.g. API systems)

The use of miniaturized biochemical systems is a standard procedure in microbiological labs. Correct identification requires the availability of a pure culture and prior determination of the cultural characteristics (gram characteristics, catalase test, oxidase test, sporulation capacity, microscopic image).

ifp uses API identification systems (Analytical Profile Index), for instance, which cover relevant groups of bacteria and yeasts. These systems determine the usability of certain carbohydrates or the formation of specific metabolic products, the evaluation of which is used to create a numerical profile. The profile is then compared to profiles in databases, which ultimately allows for identification of the microorganism. Results are ready after 4 – 72 hours, depending on which system was used.

Sequencing (16S- or 18S- or ITS-rDNA)

This molecular biological method of identification is based on multiplying a specific highly variable section of the ribosomal DNA (rDNA) of the microorganism by means of PCR (polymerase chain reaction) and subsequent sequencing of the amplificate.

The following steps need to be performed for this:

  • isolation of the genomic DNA from bacteria, yeasts or fungi (pure culture)
  • running the PCR: 16S-rDNA (bacteria) or 18S- or ITS-rDNA (yeasts and fungi)
  • purification of the PCR product
  • sequencing the PCR product
  • evaluating the sequence e.g. via NCBI BLAST

The result of the molecular biological identification will be ready after approx. 3 days.

Cultural methods (fungi)

Classic identification of fungi is based on macroscopic and microscopic analysis of pure cultures on suitable media. The morphological characteristics of the colonies and of the microscopic image are compared with data from the literature.

Microbial Identification using MALDI-TOF Mass Spectrometry

In the field of microbiology, ifp has established MALDI-TOF mass spectrometry (Matrix Assisted Laser Desorption Ionization - Time of Flight) as a method for the identification of bacteria, yeasts and moulds.

Microbial identification according to specific ribosomal protein patterns

Identification by means of MALDI-TOF relies on obtaining a unique molecular fingerprint of the respective target organism. This is done by measuring specifically the ribosomal proteins, which are found in large quantities in all microorganisms. The characteristic spectra of these proteins are used to identify a certain microorganism by comparing the respective pattern with those in several databases.

Ionization and detection in a Time-of-flight detector

To do so, the pure-culture microorganism to be identified is placed on a sample carrier either directly or after brief protein extraction, coated with an organic matrix and measured in the MALDI-TOF. Measurement is done based on laser bombardment of the analyte-matrix mixture, leading to ionization. The ions are accelerated and their specific masses are detected in the time-of-flight detector. This results in the specific mass spectrum which is used to identify the microorganism to be determined. The measuring procedure takes only a few minutes.

Advantages of microbial identification using MALDI-TOF

  • rapid and precise
  • little sample material required
  • can be used for bacteria, yeasts and fungi
  • constantly expanding database
  • readily available storage of all spectra and evaluations
  • customized databases
  • contamination traceability
  • identification of facility-specific monitoring isolates

Identification of microorganisms

Fast and safe identification of microorganisms is a prerequisite for expert evaluation and for controlling contamination in a large variety of food, cosmetic and pharmaceutical  production, and also in the context of hygiene tests. ifp Institut für Produktqualität offers the following options for identification of microorganisms:

Identification is done based on samples such as:

  • isolates, pure and mixed cultures on solid media or in liquid solutions
  • media from monitoring, such as contact plates, air samples, slides
  • plates from assays to determine microbial counts

Microorganisms can be isolated for subsequent identification from almost any type of samples from food right up to objects of daily use. Identification requires the availability of a pure culture, which is produced on a suitable medium after subcultivation.

Preservation of microorganisms

ifp Institut für Produktqualität offers long-term storage of identified isolates, for use as additional test cultures or facility-specific monitoring isolates in tests and validations or for the purpose of comparison.

Alicyclobacillus

Species of the Alicyclobacillus spp. genus are large, immobile, spore-forming, thermophilic bacteria that occur almost anywhere in the environment. The spores of Alicyclobacillus may find their way into production through contaminated fruit during harvest. They are tolerant to high pasteurizing temperatures and low pH-values, which is why they easily proliferate in juice. There are 18 known species, with Alicyclobacillus acidoterrestris being the most significant. In the event of contamination with A. acidoterrestris, specific metabolites form that produce a repulsive taste and odour but are not hazardous to health. The product itself remains visually flawless, neither gas nor discolourations form. Even small amounts of the bacteria can lead to product spoilage, which is why the beverage industry’s in-house specifications demand a zero-tolerance approach. Alicyclobacillus spp. contamination affects almost all non-alcoholic beverages, e.g. apple, pear, orange, tomato and grape juice.  


What analytical methods does ifp Institut für Produktqualität offer?

Cultures

Cultures are grown according to IFU (International Federation of Fruit Juice Producers) method no. 12 and take up to 10 days incl. confirmation in the event of a positive result. Analysis can be done quantitatively for filterable samples or qualitatively for non-filterable samples. The disadvantage of this method is that a positive result provides no information about which species of the genus was found.

PCR

ifp Institut für Produktqualität offers analysis using its own Duplex-PCR system, which is a molecular biological rapid method for the detection of Alicyclobacillus. It produces qualitative results within one to two days. This method also provides information about whether the genus found is Alicyclobacillus spp. or specifically Alicyclobacillus acidoterrestris.

 

Food Microbiology

ifp tests the hygienic and microbiological quality of food and other commodities. Along with the conventional, classical methods of microbiology we also use modern molecular biological methods such as real-time PCR (polymerase chain reaction). The corresponding test kits have been specifically developed and produced by ifp and are distributed world-wide by the company QIAGEN. The implementation of MALDI-TOF mass spectrometry enables ifp to perform rapid state-of-the-art identification of microorganisms based on a comprehensive database.

ifp has a license to work with pathogenic germs under the German Infection Protection Act.

For more information on food microbiology, see the following pages:

Excerpt from ifp's microbiological service testing portfolio

Basic parameters

  • total counts
  • yeasts
  • molds

Pathogens (excerpt)

  • Campylobacter
  • Cronobacter
  • Clostridium
  • EHEC
  • Legionella
  • Listeria
  • Salmonella

Viruses (excerpt)

  • astrovirus
  • adenovirus
  • Hepatitis A virus
  • norovirus
  • rotavirus

Spoilage bacteria (excerpt)

  • acetic acid bacteria
  • Alicyclobacillus
  • lactic bacteria
  • Pseudomonads

Hygiene indicators (excerpt)

  • Enterobacteriaceae
  • Escherichia coli

Cleaning and desinfection controls (excerpt)

  • e.g. contact plate samples
  • amount of aerobic bacteria